Approaches for determining phytoplankton nutrient limitation

Aquat.sci.63 (2001) 44– 69 1015-1621/01/010044-26 $ 1.50+0.20/0 © Birkhäuser Verlag, Basel, 2001 Aquatic Sciences Approaches for determining phytoplankton nutrient limitation John Beardall 1, *, Erica Young 2 and Simon Roberts 1 1 2 Department of Biological Sciences, Monash University, PO Box 18, Clayton, Victoria 3800, Australia School of Biology and Biochemistry, Queen’s University of Belfast, Belfast BT9 7BL, Northern Ireland, U. K. Key words: Phytoplankton, nutrient limitation, chlorophyll fluorescence, molecular diagnosis. ABSTRACT Aquatic primary productivity is frequently limited by the availability of nutrients. The ability to identify factors limiting algal growth is of considerable importance to our understanding of the ecology of aquatic plants and to water management practices. Methods used to identify limiting resources in the past have included a) analysis of nutrient availability, b) elemental composition and cell quotas for various nutrients, c) bio-assays monitoring growth of test species or of natural populations following nutrient enrichment and d) measurements of various physiological parameters, such as enhancement of respiration and dark carbon fixation rates and perturbation of photosynthetic rate following re-supply of nutrients. In this paper we briefly review the merits and methodological limitations of these approaches for the assessment of the nutrient status of algal populations. We discuss how an understanding of biochemical and metabolic changes induced by nutrient limitation has led to the development of rapid and simple tools to monitor the nutrient status of aquatic plants and algae. In particular, we describe the use of transient changes in chlorophyll a fluorescence as a potential tool for rapid assessment of algal nutrient status and the development of molecular probes specific to nutrient limited cells, such as flavodoxin as a diagnostic tool for Fe-limitation. Introduction Aquatic primary productivity, especially in surface waters, is frequently limited by the availability of nutrients. Principally this involves limitations in the supply of nitrogen and phosphate (Schindler, 1977; Wynne and Berman, 1980; Birch et al., 1981; Lean and Pick, 1981). Although N and P were originally believed to be limiting in marine and freshwater ecosystems respectively (Hecky and Kilham, 1988; but see Elser et al., 1990; Dodds et al., 1993), more recent investigations have shown * Corresponding author, e-mail: [email protected] Phytoplankton nutrient limitation 45 that some marine systems such as the North Pacific Sub-tropical Gyre and regions of the Mediterranean Sea are, in fact, P limited (Karl, 1999; Krom et al., 1991). In “high nutrient, low chlorophyll” (HNLC) areas such as the equatorial Pacific and Southern Ocean, iron has been shown to be an important limiting resource (Martin and Fitzwater 1988; Behrenfeld et al., 1996; Timmermans et al., 1998; Boyd et al., 1999), and even Zn has been implied as a limiting nutrient in some situations (Bruland, 1989; Morel et al., 1994). The identification of nutrient limitation of phytoplankton growth and the identity of the limiting factor(s) in a water body is of considerable importance to our understanding of the ecology of aquatic systems and to water management practices. It enables managers to draw up appropriate nutrient loading budgets for catchments and respond to possible perturbations on an informed basis. To have reliable and rapid techniques to do this is of considerable significance. A number of techniques have been employed in the past in order to determine factors limiting growth and production of algae. In this paper, it is our intention to review the merits and drawbacks of a number of these approaches, including some which have developed from an increasing understanding of algal metabolism under nutrient limitation. We will also review more recently developed molecular approaches and briefly describe a new, rapid and specific assay technique, based upon nutrient induced transient changes in chlorophyll a fluorescence emission from nutrient-deficient microalgae. Background – defining nutrient limitation In dealing with nutrient limitation, it is necessary to address precisely what is meant by limitation of productivity and algal growth. Many ‘bioassay’ or ‘enrichment’ experiments, used in the past to investigate the factors limiting algal growth, fail to differentiate between Liebig limitation i. e. limitation of the extent of growth (maximum yield or standing stock of algae) and the limitation of instantaneous growth rate (Fig. 1). Studies based on measurements of Liebig limitation do not necessarily reflect limitation by any particular nutrient at the time of sampling and at the prevailing cell density and species composition of the population. Rather they indicate simply that should growth continue, one particular nutrient will become the limiting factor. This distinction has major implications for the way that we approach studies of nutrient limitation in natural populations (see Cullen et al., 1992). In oligotrophic waters, a low biomass of rapidly dividing phytoplankton can be maintained by heavy grazing pressure combined with efficient recycling of nutrients in the water column. In that case, the growth rate of individual cells is not limited by nutrient availability, but the maximum population size is determined by the overall nutrient concentration. However, the questions of biomass accumulation versus rate processes may be particularly relevant with the current requirement to model and predict effects of global climate change and the role of the oceans in biological draw-down of anthropogenic CO2 (Raven and Falkowski, 1999). 46 Beardall et al. Long term manipulation and growth of phytoplankton Bioassays with ‘test organisms’ Bioassay experiments may use the growth of microalgal cells as a measure of the capacity of a water sample to support microalgal growth. Water from a given site is filtered and inoculated with a test organism. For freshwaters, organisms such as Selenastrum capricornutum have been used (EPA, 1971; Miller et al., 1974, 1978) and other species including Thalassiosira pseudonana have been used in marine samples (Hayes et al., 1984). Growth of the test organism is measured in the presence of specific added nutrients, and if addition of a particular nutrient leads to enhanced growth, it is deduced that that nutrient was limiting in the original sample (Fig. 1). This type of assay system is easy to set up and measurements of biomass over time are relatively simple to make, rendering them attractive in laboratories with fewer resources, and in the field. Nonetheless, there are some inherent problems with this approach. Ideally, the growth rate is the measured parameter. Frequently, however, bioassays rely on final biomass (yield, Y in Fig. 1) attained after a period of many days i.e. they are essentially measures of Liebig limitation. This simply provides information about the maximum biomass that the water body could sustain, and which nutrient will first become limiting as the population increases. These type of assay systems also tend to reflect the nutrient requirements for growth of one specific test organism, which may bear little resemblance to the responses of the species characteristically found in the water body under investigation. Filtration of Figure 1. A stylised representation of a growth bioassay. Assuming N limitation in the original sample (control), addition of P elicits no change in growth rate (µ, as represented by the slope of the Log [biomass] vs time plot) or final yield (Y). Addition of N, however, can cause an increase in µ. At the same time, the added nutrient allows an increase in population size so the final yield YN is larger. With added N, P can become the limiting nutrient at high biomass levels so addition of N + P can allow further increases in population size to YN + P Phytoplankton nutrient limitation 47 the water sample prior to inoculation with the test organism may also remove colloids and organic complexes that are frequently a source of nutrients, especially phosphorus (Wood and Oliver, 1995). Enrichment experiments with natural populations More commonly, enrichment experiments have been carried out with natural water samples, relying upon the initial natural phytoplankton population as inoculum. A sample containing a natural population is “spiked” with various nutrients or with a nutrient mix lacking one particular nutrient. Carbon assimilation (Menzel and Ryther 1961; Beardall et al., 1982) or growth (Cullen et al., 1992; Dodds et al., 1993) or the variable chlorophyll a fluorescence parameter Fv/Fm (Kolber et al. 1994; see below) is then determined over a period of time varying from several hours to several weeks or even longer (Healey, 1979; Hecky and Kilham, 1988). If productivity (14C fixation or photosynthetic O2 evolution) or growth (measured as changes in biomass from chlorophyll concentration or cell numbers) is similar in samples without a particular nutrient and in a control with all tested nutrient additions, it is usually deduced that the nutrient is not limiting. On the other hand, if a particular nutrient addition stimulates production and/or growth, that is indicative of that nutrient being a limiting factor in the original water mass. As with bioassays based on test organisms, experiments involving enrichment of natural phytoplankton populations are not without problems (see Esler et al., 1990). For instance, the response to bioassay enrichment experiments may be influenced by the species composition of the seeding/inoculum phytoplankton sample and addition of one macronutrient may induce limitation of other nutrients. For instance, Menzel and Rhyther (1961) demonstrated a stimulatory effect of iron enrichment on 14C uptake by phytoplankton samples from the Sargasso Sea over 24 h. However, over a longer period of 3 days, N and P additions were needed to produce a comparable stimulation. Chemical speciation is important and, especially with trace metals, contamination and chelation effects can be significant (see Butler, 1998). Assessment of micronutrient deprivation on the basis of concentrations available in the water column requires trace-metal-clean methods for sampling water and relatively sophisticated analytical methods (Bruland, 1989; Twiss et al., 2000). Chemical speciation of transition metals, and organic complexation of trace metals within the water column (particularly in freshwater environments, where the organic load, including humic substances, may be higher) complicates the relationship between measured concentrations and bioavailability for phytoplankton uptake (see Anderson and Morel, 1982; Butler, 1998; McKay et al., unpublished). The complexity of trace metal chemistry has important implications both for interpretation of micronutrient availability as well as for enrichment bioassays, where alleviation of micronutrient limitation has commonly been achieved by addition of iron together with a chelating agent (usually EDTA) (e.g. Menzel and Rhyther, 1961; Peeters and Peperzak, 1990; Boyd et al., 1998). Furthermore, Muggli and Harrison (1996) showed the potential for inhibitory effects of EDTA on growth of phytoplankton. In enrichment bioassays, it can also be difficult to resolve trace metal limitation as the alleviation of one deficiency pre- 48 Beardall et al. cipitates another eg. alleviation of macronutrient limitation induces micronutrient deficiency. In addition, physical enclosure of a natural population can have deleterious effects upon physiological performance and result in changes in species composition of the phytoplankton, and the population can be isolated from important nutrient sources such as sediments, particulate matter or recycling (Venrick et al., 1977; Healey, 1979). Removal of zooplankton can also cause a relief from grazing pressure that can result in significant stimulation of growth even in unenriched control bottles (Cullen et al., 1992). If population growth is thus stimulated, the biomass may increase until the availability of some nutrient eventually becomes limiting (i.e. a Liebig limitation eventually sets in). Furthermore, if samples are incubated at a higher photon flux than they might experience in situ, populations that were originally light-limited are exposed to conditions in which that limitation is alleviated, allowing a potential limitation by some nutrient to be expressed. Results obtained from such experiments may bear little resemblance to the nutrient status of the original plankton assemblage. Furthermore, in enrichment bioassays based on short term 14C fixation or O2 exchange, addition of the limiting nutrient can actually cause short-term decreases in the rate of photosynthesis (see Elrifi and Turpin, 1987) which are related to interactions between nutrient uptake and assimilation and other metabolic processes (see below). An important development in the bio-assay approach to assessing nutrient limitation is the recent use of large-scale in situ enrichment experiments. These have been exemplified by the increasing number of Fe-enrichment trials, which have identified Fe-limitation in “high nutrient-low chlorophyll” (HNLC) regions of open ocean. These Fe enrichment experiments will be mentioned below repeatedly in the context of specific parameters, but for a review of the general design of such experiments see Coale et al. (1998). More immediate indices of nutrient status Elemental ratios and macromolecular composition Since the seminal work of Redfield (1958), the elemental composition of phytoplankton, and even the composition of the water in which the organisms are growing, has been used as a potential index of nutrient limitation. Phytoplankton cells/populations on average assimilate C, N and P in the approximate ratio 106: 16:1 (the Redfield ratio), so a water body with a N:P ratio of 30 is likely to lead to P limitation of algal growth whereas a ratio of 5 would imply N limited growth; in Scenedesmus sp., P limitation switches to N limitation when the cellular N:P ratio is about 30 (Rhee, 1974). Rees et al. (1995) measured the C:N assimilation ratio in a field study and used this to assess N limitation during the development of a spring phytoplankton bloom. The use of elemental ratios of a water body can provide evidence for possible Liebig limitation of algal populations but are less useful for ascertaining whether a given phytoplankton population is nutrient limited at a given point in time. Furthermore, the reliance on Redfield ratios has been questioned by Phytoplankton nutrient limitation 49 Falkowski (2000) who pointed out that the ratio is far from constant and has been embraced by aquatic ecologists as conveying more information than is warranted. This is further complicated by the difficulties in determining the availability of nutrients for algal growth from a water sample, especially in turbid systems with high levels of particulates (White and Payne, 1980; White et al., 1985; Wood and Oliver, 1995). Methods for extracting phosphorus, for example, are complex and may not reflect the availability of that element to phytoplankton (Bostrom et al., 1988); measurements of particle-associated exchangeable phosphorus show much better correlation to growth bioassays than does total dissolved phosphorus (Oliver, 1993). The use of nutrient concentrations prevailing in a particular water body to determine which nutrient is limiting also does not account for regeneration of nutrients within surface waters (which can vary significantly between water bodies), the autotrophic and heterotrophic community structure, and inorganic and organic nutrient inputs (i. e. variable f ratios) (Dugdale and Goering, 1967; Eppley and Peterson, 1979). In addition to concentrations within the water column, assessment of the pool size and rate of turnover of a particular nutrient is needed to assess (potential) limitation (Harris, 1986). Sas (1989) has demonstrated the importance of sediment in supporting phytoplankton growth, where there have been large reductions in phosphorus load in the water column. The control of phytoplankton growth rate through other factors such as light may also complicate interpretation of elemental ratios for assessing nutrient status (see Goldman et al., 1979; Laws and Bannister, 1980; Tett et al., 1985), though growth at low light per se was not found to influence particulate C:N:P ratios in Nlimited cultures of the chrysophyte Pavlova lutheri (Goldman, 1986) or C:N ratios in the diatom Thalassiosira pseudonana (Thompson, 1999). The elemental composition of algae reflects the macromolecular composition (protein, carbohydrate/lipid) composition of cells, which in turn reflects the integration of the various processes involved in nutrient uptake and assimilation. Elemental ratios thereby provide an integrated measure of an algal cell’s more immediate nutrient history (Goldman et al., 1979; Healey, 1979; Fresnedo and Serra, 1992). Thus nitrogen limitation usually results in diminished protein content and relatively enhanced carbohydrate or lipid storage (Morris, 1981; Shifrin and Chisholm, 1981; Ganf et al., 1986) while P limitation can also cause a shift in the proportions of protein, lipid and carbohydrate (Harris and Piccinin, 1983; Harrison et al., 1990; Theodorou et al., 1991; Reitan et al., 1994). Unfortunately, changes in elemental ratios of algal samples are not necessarily specific to the limiting nutrient and C:N ratios and N:P ratios can be influenced not only by N limitation but also by Si limitation or inorganic carbon availability (Table 1). In addition, measurements of elemental ratios or chemical composition of natural populations of phytoplankton are prone to interference from debris and other microorganisms such as bacteria and microzooplankton that can be collected on the filters used to sample the phytoplankton. One possible way to avoid interference from other organisms and debris in estimations of cell composition is the application of Fourier Transform Infrared (FTIR) spectroscopy. In combination with microscopy, this technique can be used to determine macromolecular content of individual algal cells or colonies, thereby avoiding any signal from debris or other particles (see Beardall et al., 2001 – this issue). However, the changes in macromolecular composition of cells brought 50 Beardall et al. Table 1. Representative values of C: N: P ratios of microalgae under different conditions. The C:N:P ratio of microalgae is not a useful indicator of the limiting nutrient. Both Si limitation and CO2 availability can influence these ratios. C: N ratios can be influenced by P status and vice versa Nutrient status REDFIELD C: P 106 C: N 6.6 Species Si (starved) N (starved) 104 62 13.2 14.8 Skeletonema (Bacillariophyceae) P limited (µ = 0.041 h–1) P limited (µ = 0.017 h–1) N limited (µ = 0.041 h–1) N limited (µ = 0.017 h–1) N limited (µ = 0.0085 h–1) 220 665 63.4 84.3 90.5 5.8 10.7 5.7 10.0 14.8 P limited 10% µmax P limited 50% µmax N limited 10% µmax N limited 50% µmax 600 325 160 120 12.5 10.2 16 12 CO2 = 180 ppm CO2 = 540 ppm 64.5 72.7 CO2 = 350 ppm CO2 = 3500 ppm 250 490 CO2 = 350 ppm CO2 = 1000 ppm CO2 = 350 ppm CO2 = 1000 ppm 115.5 128.7 109.5 120.5 6.3 6.2 n/m n/m 6.1 5.7 5.9 5.9 Reference Harrison et al. 冧 1977 冧 Perry 1976 Dunaliella (Chlorophyceae) 冧 Goldman et al. 1979 Skeletonema (Bacillariophyceae) Burkhardt 冧 and Riebesell 1998 Ochromonas (Chrysophyceae) 冧 Caraco et al. 1996 Chaetoceros (Bacillariophyceae) Dunaliella tertiolecta (Chlorophyceae) 冧 Jenkins and Beardall (unpublished) Thalassiosira (Bacillariophyceae) about by nutrient limitation are, as stated above, not sufficiently specific to allow FTIR techniques to identify which nutrient is limiting in a given situation. Interactions between nutrient status and algal metabolism Nutrient Uptake Kinetics Nutrient limitation of algal growth results in changes to algal metabolism that can be used as a measure of nutrient status. Given the extended time necessary for conducting bioassays and the additional problems, outlined above, with these and other parameters such as elemental ratios, physiological analyses offer the possibility of more rapid estimates of nutrient status. The first step in assimilation of nutrients into organic matter is their uptake into algal cells and there have been numerous studies investigating the effects of nutrient status on kinetics of nutrient uptake. In natural waters the concentration of two of the major nutrients, nitrogen and phosphorus, are often very low in comparison to measured values of half saturation constants (K m) for the inorganic ions (Harrison et al., 1996). It is well established that algae respond to nutrient limitation by increasing uptake capacity and/or efficiency for the specific nutrient. For example, Gotham and Phytoplankton nutrient limitation 51 Rhee (1981) demonstrated that the maximal rate of phosphate uptake, for a range of freshwater cyanobacteria and microalgae, was enhanced as P-limited growth rate decreased, and Graziano et al. (1996) reported that maximal uptake rates of Dunaliella tertiolecta under P-limitation were an order of magnitude greater than in P-replete cells. In most cases, increased uptake rate is associated with an increase in porter density (uptake sites) on the plasmalemma, rather than induction of isozymes with different kinetic properties, so the affinity of uptake for P, as reflected in half-saturation constants, is unchanged (Graziano et al., 1996; Donald et al., 1997). The utilization of the inorganic nitrogen sources, nitrate, nitrite and ammonium, by phytoplankton in field conditions has been subject to intensive study, and the organic N sources urea, amino acids and even purines and pyrimidines have been shown to support microalgal growth (Syrett, 1981). The kinetics of N uptake in relation to nitrogen deficiency is well characterised in a range of laboratory cultures of phytoplankton species grown under both steady-state N limitation and N depletion (to stimulate a starvation condition) (reviewed by Collos and Slawyk, 1980; Goldman and Glibert, 1983). N or P-deficient cells exhibit rapid uptake rates of the limiting nutrient immediately after it is re-supplied (Goldman and Glibert, 1983; Parslow et al., 1984b; Cochlan and Harrison, 1991). The maximal rate of uptake under these conditions is significantly greater than the rate required to maintain the growth of the organism at its maximal rate (Parslow et al., 1984 b) i. e. the cells exhibit “luxury uptake”. This uncoupling between nutrient uptake and growth rates increases as N deficiency becomes more severe. Transient uptake rates vary with species, nitrogen history and nutrient status (Parslow et al., 1984a; Collos, 1980) although considerable species differences have been observed in the way N deficiency influences nitrate or ammonium uptake (e. g. Dortch et al., 1982). Furthermore, the determination of uptake rates can be complicated by a range of factors including incubation conditions and time, non-stable substrates, recent presence of alternative N sources (NO –3 vs NH +4 ) and different cellular N status (Flynn, 1998). It has been established that the presence of ammonium can decrease nitrate uptake and/or assimilation capacity (e. g. Dortch et al., 1982, 1991) and, although the nature of this interaction is far from ubiquitous (e.g. Dortch, 1990), interactions between NO –3 and NH +4 sources for phytoplankton uptake have been observed in situ (e.g. Harrison et al., 1996). Secondary effects of uptake and incorporation The uptake and assimilation of N and P both require the expenditure of energy as ATP and/or reducing equivalents (Fig. 3). Many of the physiological parameters that are used as indices of nutrient status are based on changes in cell function that occur during nutrient limitation and/or on the perturbations in energy metabolism that occur when a limiting nutrient is re-supplied to cells. The ATP content of algal cells decreases during the onset of P or N deficiency and increases over a period of days following re-supply of the limiting nutrient (Healey, 1979). Re-supply of a limiting nutrient leads to ‘surge’ uptake of that nutrient (Parslow et al., 1984 a; see above). Consequently ATP derived from the light reactions of photosynthesis can be used for rapid uptake of the nutrient at the expense of C assimilation (Fig. 3). If 52 Beardall et al. the ATP is derived from enhanced cyclic photophosphorylation, rather than linear electron transport, photosynthetic O2 evolution will be diminished while nutrient is being taken up by the cells (Healey, 1979; Roberts, 1997; see also Beardall et al., 2001). The combination of enhanced nutrient uptake by nutrient limited cells, reduced O2 evolution and C assimilation rates and enhanced CO2 evolution rate following nutrient re-supply (Lean and Pick, 1981; Lean et al., 1982; Birch et al., 1986; Goldman and Dennett, 1986; Istvanovics et al., 1992) led Lean and Pick (1981) to use the ratio of C fixation (or O2 evolution) to nutrient uptake rate as an index of limitation for natural populations of lake phytoplankton. Ratios above 100 were taken as indicative of nutrient sufficiency while lower ratios suggested different degrees of limitation (Table 2). This index compared well to other parameters such as turnover time for phosphate (Lean and Pick, 1981). Similar observations on the suppression of photosynthetic C fixation were recorded by Falkowski and Stone (1975) following NO –3 and NH +4 additions to Nstressed phytoplankton cells, which they interpreted as a competition between N assimilation and C fixation for ATP (Fig. 3). Such observations of short-term inhibition of C assimilation or O2 evolution confound interpretation of short-term enrichment experiments based on changes in 14C assimilation (see above). Morris et al. (1971) first noted that N limited phytoplankton showed enhanced rates of carbon assimilation in the dark, and changes in net CO2 and O2 exchange. These phenomena have provided useful indices of N-limitation (Vincent, 1981; White et al., 1985; Elrifi and Turpin, 1987) but it was not until the elegant series of studies by Turpin and co-workers (see Huppe and Turpin, 1994 for a summary) that the biochemical basis of these observations was fully comprehensible. In brief, resupply of NO –3 or NH +4 to N-stressed cells leads to a stimulation of inorganic N assimilation and amino acid biosynthesis. To satisfy the demand for C– skeletons to support ammonium assimilation to glutamate, glycolysis and mitochondrial respiration are stimulated. Carbon to support this respiratory activity is diverted from the chloroplast, via the triose-phosphate shuttle, and is also supplemented by anaplerotic carbon fixation via b-carboxylation. Since b-carboxylation is light independent, this is the basis for the observed enhancement of dark C fixation following Naddition. Export of triose sugars from the chloroplast depletes Calvin cycle C3 intermediates and inhibits recycling of the C acceptor RuBP. This results in a reduction Table 2. Scale of P-limitation derived from the ratio of C assimilation to phosphate uptake capacity (mol mol–1) in natural populations of phytoplankton from North American lakes (from Lean and Pick 1981). This scale of nutrient deficiency correlates with other estimates, based on measurements such as P-turnover times in the populations Nutrient status Molar ratio of C assimilation/ phosphate uptake capacity Replete Low deficiency Moderate deficiency Extreme nutrient stress > 100 30 – 100 10 – 30 < 10 Phytoplankton nutrient limitation 53 in Calvin cycle activity which affects the regeneration of electron acceptors (e. g. NADP+) that are necessary to maintain linear electron flow and photosynthetic O2 evolution. This dynamic also provides a rationale for the observed inhibition of O2 evolution following N re-supply to N-limited cells (see also Elrifi and Turpin, 1987). Biochemical and Molecular approaches A large body of research has characterized the physiological, biochemical and molecular changes associated with acclimation of microalgal cells to nutrient stress. Although this work has predominantly concentrated upon a few key species, manipulated under laboratory culture conditions, it provides an essential theoretical background to nutrient stress effects on algal metabolism, and has thus characterised changes which can be applied as diagnostic tools for assessing nutrient limitation in natural phytoplankton populations. The parameters discussed in this section have in most cases been reported in combination with additional physiological assessment of algal photosynthetic competence, discussed in part above, though in recent years measures of photochemical efficiency (see section on chlorophyll a fluorescence below) have become more common. The impact of macronutrient and Fe deprivation on cellular pigment ratios has been reported in a number of phytoplankton taxa (e. g. Falkowski et al., 1989; Herzig and Falkowski 1989; Cleveland and Perry, 1987; Sosik and Mitchell, 1991; Greene et al., 1992; Geider et al., 1993, 1998; van Leeuwe and Stefels, 1998). Under N and P limitation, cellular chlorophyll content, and hence chl a:C ratio, decreases (e.g. Geider et al., 1998), though P limitation can provoke less rapid responses than Fe or N stress in some species (see La Roche et al., 1993). Geider et al. (1993) showed changes in chl c:chl a ratios in the diatom Phaeodactylum tricornutum under N, P and Fe deprivation, but found no significant changes in chl a:chl b ratio in Nlimited cells of the chlorophyte Dunaliella tertiolecta (Geider et al., 1998), although in N-starved D. tertiolecta, chlorophyll b was preferentially lost over chlorophyll a (Young, 1999). Generally, N deprivation affects chlorophyll more strongly than carotenoids, with elevated carotenoid to chlorophyll ratios a feature of N-stressed cells (e.g. Cleveland and Perry 1987; Falkowski et al., 1989; Herzig and Falkowski 1989; Sosik and Mitchell, 1991). Heath et al. (1990) applied light absorption ratios at 480 nm versus 665 nm to quantify the carotenoid:chlorophyll ratio as a potential rapid index of phytoplankton nutritional status. They found absorption ratios strongly correlated with C:N ratio, independent of light and temperature effects, but that these ratios varied between species. Although inter-specific differences were less than between nutrient replete and nutrient stressed populations, field trials suggested that for mixed populations under mild N stress, this may not be as clear a diagnostic tool as desired. Geider et al. (1998) have more recently demonstrated complex interactions between the different carotenoid pools in D. tertiolecta in response to N and P stress. Furthermore, changes in pigment content, ratios and distribution under N, P or Fe deprivation are related to changes in absorption cross section areas of Photosystem II (PSII) and chlorophyll-specific absorption which can change during N, P or Fe limitation (Herzig and Falkowski, 1989; Greene et al., 1992; Geider et al., 1993). 54 Beardall et al. Changes in Proteins An important technique with diagnostic potential for assessing nutrient deprivation in phytoplankton is characterization of cell protein profiles. Single dimensional or 2-dimensional polyacrylamide gel electrophoresis separates and resolves total proteins or polypeptides and the relative abundance and presence of particular protein bands in response to nutrient deprivation can be identified. Several authors have shown changes in ratios between relatively abundant chloroplast proteins such as the large and small sub-units of Rubisco (LSU/SSU), the PSII reaction centre protein D1, and light-harvesting chlorophyll-binding proteins (LHCI/II) under nutrient stress (e.g. Falkowski et al., 1989; Geider et al., 1993, 1998; La Roche et al., 1993; Graziano et al., 1996). Nitrogen deprivation in microalgae generally results in a preferential repression of chloroplast protein synthesis (Plumley and Schmidt, 1989). A suite of specific proteins have been identified in a range of species as being synthesised or lost in response to nutrient deficiency (e. g. La Roche et al., 1993; Li et al., 1998), yet relatively few have as yet been characterised to the extent of being useful as a priori markers for physiological limitation by nutrient stress. A specific membrane protein (nrp1), identified in Emiliania huxleyi as regulated by inorganic nitrogen limitation, was identified by Palenik and Koke (1995) using a biotinylated marker. In Synechococcus, novel membrane polypeptides have been identified under Fe, P and Mg limitation conditions (Scanlan et al., 1989) and a Synechococcus protein (PstS), induced under P-stress, showed homology with a periplasmic phosphate-binding protein of E. coli (Scanlan et al., 1993; Carr and Mann, 1994). Graziano et al. (1996) reported that abundance of a unique stress protein expressed under P stress in D. tertiolecta correlated with rising C:P ratios and elevated Vmax for phosphate uptake induced under increasing phosphate limitation. La Roche et al. (1993) provided further evidence that induction of specific proteins indicated nutrient-limitation, but that the presence of these novel proteins is less useful for determining which nutrient is limiting in mixed assemblages. Diagnosing nutrient limitation on the basis of bands on a gel is rather tenuous. However the identification and characterization of nutrient stress proteins in phytoplankton potentially enables highly specific immunological detection of nutrient stress markers, as the successful identification of Fe-stress proteins in diatoms attests (see section below). Dortch et al. (1985) reviewed a range of biochemical indices of nitrogen deficiency in natural phytoplankton populations, including deducing nitrogen sufficiency from high amino acid/protein ratios. They discussed variations in inorganic nitrogen storage, enzyme activity and protein/DNA ratios and their utility in understanding nutrient deficiency in phytoplankton. Specific enzyme markers for nutrient limitation Alkaline Phosphatase Extracellular alkaline phosphatase (AP) is expressed in a large range of phytoplankton taxa in response to P-limitation. AP is highly stable in seawater, and thus Phytoplankton nutrient limitation 55 has potential as a indicator of inorganic P limitation, though it may reflect recent P status, rather than the prevailing P status. Alkaline phosphatase catalyses the breakdown of exogenous organic-P compounds to yield utilizable inorganic P forms. The appearance of AP as an index of P-limitation was first proposed by Reichart et al. (1967) and extensively applied in freshwater systems by Berman (1970). Alkaline phosphatase activity (APA) has been detected in P-limited but not in nutrientreplete or N-limited phytoplankton (e. g. Sakshaug et al., 1984; Dyhrman and Palenik, 1997), and in freshwaters APA has been associated with orthophosphate depletion during spring phytoplankton blooms (Stevens and Parr, 1977). Significant APA has been shown to correlate well with chlorophyll concentration during freshwater phytoplankton blooms, and also correlated well with indices of P limitation (bioassay, C:P ratios) and depletion of soluble reactive phosphorus (SRP) concentration in the water column (Vrba et al., 1995; Rose and Axler, 1998). Rose and Axler (1998) showed a reduction in APA with P enrichment, whilst other nutrient additions increased activity. APA is thus apparently a good index of phytoplankton P status, though Rose and Axler (1998) recommended caution in interpreting APA data in isolation as, when N-P co-limitation or secondary N limitation is present, APA activity may indicate P limitation without corresponding growth increases occurring under P enrichment. This re-iterates the strength of multiple assessment methods used simultaneously. Detection of significant APA may require relatively severe P limitation in some species. Graziano et al. (1996) showed APA in severely P-starved D. tertiolecta whilst APA was not detected in P-limited cells. In contrast, a distinctive protein marker of P-stress in D. tertiolecta was detected shortly after the onset of P-starvation (Graziano et al., 1996). APA detected in water samples can include a contribution from bacterial enzymes, though size-fractionation of water samples can reduce this contamination. APA can also be released from cells and be found dissolved in water samples. The relationship between APA and the degree of P stress varies amongst species (Graziano et al., 1996 and refs therein), and there may be a diversity of P-limitation within mixed phytoplankton assemblages. Li et al. (1998) investigated the spatial distribution of APA in different plankton size fractions and in aqueous fractions from the Red Sea. The majority of particulateassociated APA was in the picoplanktonic fraction, and there was significant correlation of APA with Synechococcus numbers, implying that APA responses to Plimitation may depend on species composition. González-Gil et al. (1998) recently reported a new in situ assay for APA in single cells, for detection by microscopy or in combination with flow cytometry and this may prove a useful advance in understanding the nutrient status of individual species within a mixed population (see also Zettler et al., 1996). Enzymes of Nitrogen Assimilation Fewer studies have looked at enzymes of nitrogen assimilation as indices of nutrient limitation, though changes in activities of these enzymes in relation to N-limitation, N-starvation and interactive effects of nitrogen source (NH +4 vs NO –3) are well understood (e. g. Goldman and Glibert, 1983). Nonetheless, activities of several enzymes associated with N-assimilation, viz. nitrate reductase (NR), glutamine syn- 56 Beardall et al. thetase (GS) and NADPH-glutamate dehydrogenase (GDH) have been investigated as possible indicators of N-limitation (Dortch et al., 1979). Changes in GS and GDH activities have been associated with increasing N-limitation in (chemostatgrown) Chlorella stigmatophora (Everest et al., 1986). NR activity has been measured in concert with 15N uptake, 14C fixation, pigment concentration and particulate N, and related to the N concentration in the water column in marine systems (Blasco and Packard, 1974; Packard et al., 1978). Wynne et al. (1990) examined NR activity together with nitrate and ammonium uptake capacity and cellular N:P ratios in freshwater phytoplankton, but suggested that NR activity was related to nutritional N status and nutrient pre-history rather than ambient conditions. Furthermore, conflicting responses to N-depletion have been observed in a range of marine microalgae with NR activity increasing (e. g. Kessler and Osterheld, 1970; Watt et al., 1992) or decreasing (Eppley et al., 1969, Berges et al., 1995) as depletion progressed. Consequently, measurements of levels of enzymes of nitrogen assimilation are probably unlikely, in isolation, to provide a definitive tool for detecting Nlimitation in phytoplankton populations. Ferredoxin – Flavodoxin Since the proposal that oceanic productivity can be limited by iron availability in high nutrient/low chlorophyll (HNLC) regions of the open ocean (Martin and Fitzwater, 1988), there has been strong interest in assessing physiological Fe limitation of phytoplankton, with the redox catalysts ferredoxin and flavodoxin emerging as promising candidates for a specific assay. Ferredoxin (Fd) is a highly conserved non-haem iron-sulphur protein which plays a central role in energy metabolism of cells by mediating electron transfer. A response common to prokaryotic organisms and eukaryotic phytoplankton subjected to iron-stress is the substitution of Fd with flavodoxin (Flv), a non-iron containing functional equivalent (La Roche et al., 1993). Although some prokaryotic and eukaryotic algae produce trace levels of Flv consitutively (see Doucette et al., 1996), elevated expression of Flv in a range of diatoms species tested is induced by iron-stress, leading La Roche et al. (1993) to suggest that Fd and Flv can be used as molecular probes of Fe-stress in the ocean. Flavodoxin induction is also found in members of the Prymnesiophyceae and Dinophyceae (Erdner et al., 1999) and the cyanobacterium Nostoc (Hutber et al., 1977). The detection of Fd and Flv in phytoplankton has utilised predominantly immunological techniques (La Roche et al., 1993, 1995, 1996; McKay et al., 1997, 1999; Timmermans et al., 1998), but also FPLC, and HPLC (Doucette et al., 1996 and references therein; Erdner and Anderson, 1999). In further characterising the broader potential for immunodetection of Fd/Flv expression to diagnose Fe-limitation in phytoplankton, McKay et al. (1999) detected Fd in a range of microalgae using antibodies generated against Fd purified from the diatom Thalassiosira weissflogii. Abundant Fd was also detected by HPLC in Fe-replete cells of T. weissflogii but undetectable in Fe-limited cells (Doucette et al., 1996). McKay and co-workers (1999) showed that flavodoxin accumulation varied inversely with levels of ferredoxin, which was not detectable in cells of the Fe-deficient diatom Phaeodactylum tricornutum. Erdner and co-workers proposed an Fd abundance ratio (Fd/(Fd + Phytoplankton nutrient limitation 57 Flv)) as a quantitative index of Fe stress (Doucette et al., 1996; Erdner and Anderson, 1999). Relative Fd and Flv can be related (McKay et al., 1999) to a frequently used parameter of physiological limitation, Fv/Fm (see below). However McKay et al. (1997) also showed that Flv can be expressed in the early stages of Fe-limitation and may be independent of growth rate and other indices of physiological limitation by Fe in both P. tricornutum and T. weissflogii. Whilst most reports of Fd/Flv expression have concentrated on a few key diatom species in laboratory culture, the variable abundance of ferredoxin and flavodoxin in response to Fe stress has been demonstrated in field populations, in context with in situ oceanic Fe-enrichment experiments (La Roche et al., 1995, 1996; Timmermans et al., 1998; Erdner and Anderson, 1999) and in freshwater systems (McKay et al., unpublished). La Roche et al. (1996) reported detection of flavodoxin in phytoplankton from HNLC regions but not from high iron regions of the sub-arctic Pacific. Furthermore, relative flavodoxin abundance declined over 6 days following Fe enrichment despite large concurrent increases in chlorophyll, and Flv could be specifically localised to chloroplasts of single diatom cells using immunofluorescence microscopy (La Roche et al., 1996). In Fe-enrichment experiments, diatoms apparently respond most dramatically to Fe additions, which requires caution for interpreting Flv and Fd data. Timmermans et al. (1998) showed an apparent increase in Flv abundance following minimal Fe enrichment in bottle experiments. However, in their experiments, there was a shift in species composition towards diatoms, and the incomplete alleviation of Fe deficiency resulted in an increase in Flv, in the increasing diatom population, relative to a total protein marker. An important control for immunodetection of any specific protein marker is calibration against total algal protein, or comparison with a non-specific immunomarker, typically Rubisco LS (e.g. McKay et al., 1999). Immunodetection of Fd and Flv provides a very sensitive tool for assessing Fe deficiency. However, limited antibody cross-reactivity allows restricted taxonomic representation in reports of Flv expression under Fe stress (see McKay et al., 1999), which has also biased existing studies towards diatoms. Diatoms are major taxa of ecological relevance which are particularly iron-sensitive, however additional antisera would could allow Flv /Fd expression to be characterized in a wider range of phytoplankters, and thereby broaden and strengthen Flv/Fd abundance as a diagnostic for Fe deficiency. Detection of Flv and Fd with HPLC (Doucette et al., 1998; Erdner and Anderson, 1999) avoids the phylogenic bias but is less sensitive than immunodetection, so requires a larger volume of cells, which could be difficult to achieve from HNLC areas of open ocean. Other metal-containing enzymes can also potentially be used as markers for trace metal deficiency. The role of trace metals in algal photosynthesis has been recently reviewed by Raven et al. (1999). Antisera generated against the Mn- and Fe-containing isoforms of superoxide dismutase (Matta et al., 1992) could provide an assay for Mn deficiency and an additional marker for Fe stress. The Cu-containing redox catalyst plastocyanin is similarly replaced with a non-Cu containing compound in some picoplankton species that may become Cu deficient in freshwaters where organic complexation may limit Cu bioavailability (Arudchandran and Bullerjahn, 1996). 58 Beardall et al. Nucleic Acids RNA/DNA ratios and DNA/Carbon ratios have been briefly examined to a limited extent for their potential to estimate amount of living material in a natural phytoplankton assemblage (e.g. Dortch et al., 1985). This technique has a major limitation in application to a mixed population, in that the pools of C and of nucleic acids can be contributed inequitably from different taxa, so changing ratios could represent changing species composition rather than a response to nutrient stress. However nucleic acid ratios could potentially be used as an adjunct to estimating growth from algal yield in bioassay style experiments. The use of PCR technology may enable detection of specific mRNA species associated with nutrient-regulated genes (e.g. see Leonhardt and Straus, 1992). A problem common to assessment of nutrient deficiency using elemental analyses and enzyme activities and other physiological/biochemical markers is interference or uncertainty associated with presence of non-photoautotrophic organisms (microzooplankton, bacteria, viruses) and in phylogenetic variation. This source of ambiguity is not associated with use of methods specific to photosynthetic function i.e. measuring productivity or chlorophyll (spectrophotometric or fluorescence), or growth characteristics with chlorophyll as a biomass determinant, or to the targeting of specific algal taxa made possible with some immunological and molecular approaches (see above). Chlorophyll a Fluorescence Light energy absorbed by chlorophyll in the light-harvesting antennae of the chloroplast thylakoid membranes has 3 major fates (Fig. 2). Most of the absorbed energy is re-emitted as heat, some can be used to do photochemical work in driving ATP synthesis and NADP+ reduction, and a proportion is re-emitted as fluorescence. These pathways compete for the light energy absorbed. Thus the proportion of energy used to do photochemical work is inversely related to the amount of fluorescence emission from chlorophyll a. The capacity for photochemical work is influenced by the nutrient status of cells and any damage to the photochemical apparatus caused by photoinhibition. The instantaneous capacity for photochemical work also depends upon reactions in the chloroplast, which utilize photosyntheticallyderived ATP and reductant. Changes in rates of photochemical processes are thus reflected in changes in fluorescence output. Fluorescence therefore provides an extremely sensitive tool for examining energy metabolism in photosynthetic cells and the interactions between processes such as C assimilation and nutrient assimilation that compete for electrons derived from the H2O-splitting reactions in PSII. Chlorophyll a fluorescence can be readily measured by a range of fluorometers, provides a non-invasive assessment of the efficiency of photochemical conversion by PSII, and has become an increasingly powerful tool for investigations of nutrient limitation in phytoplankton. The fluorescence parameter most commonly used is the variable fluorescence expressed as Fv /Fm: Fv/Fm = (Fm – F0)/Fm Phytoplankton nutrient limitation 59 Figure 2. The fate of light energy absorbed by chlorophyll in algal cells. Light absorption causes excitation of a chlorophyll molecule when an electron in the chlorophyll molecule is lifted to a high energy level. As this electron returns to the low energy ground state, it releases energy, which can be given off as heat or as fluorescence or can be used to power chemical work such as the assimilatory processes shown. Clearly, these demands on the excitation energy are competitive and changes in assimilation rates will be reflected in opposing changes in fluorescence or heat output where Fm is the maximum fluorescence from a sample (i. e. when capacity for chemical work is saturated) and Fv is the difference between Fm and the minimum fluorescence F0 , obtained after a period of dark adaptation, when the capacity for chemical work is maximal. For details of fluorescence and its use in measuring photochemical efficiency the reader is referred to Maxwell and Johnson (2000). Although many early studies obtained Fm values by blocking electron transport with the inhibitor DCMU (e.g. Geider et al., 1993), many investigations now use either Fast Repetition Rate (FRR; Kolber et al., 1988) or pulse amplitude modulated (PAM) fluorometers (Schreiber, 1994). In these systems, Fm is obtained following an intense light flash which rapidly saturates the photochemical apparatus. Measurements of variable fluorescence Variable fluorescence is extensively used to monitor effects of environmental stress on algal photosynthesis and applications include phytoplankton nutrient deficiency. The photosynthetic light harvesting and energy transduction apparatus is biochemically rich in N and Fe, and P plays a crucial role in cellular metabolism (as a component of membranes) and in energy transduction (as adenine nucleotide phosphates). Thus deficiency in these nutrients result in major impairment of photochemical efficiency, Fv/Fm . Decreases in Fv/Fm have been observed during N-, P- or Fe-starvation in a range of algal taxa (e. g. Kolber et al., 1988; Herzig and Falkowski, 1989; Sosik and Mitchell, 1991; Geider et al., 1993, 1998; La Roche et al., 1993; Greene et al., 1992; Berges et al., 1996; McKay et al., 1997; Young, 1999). Changes in variable 60 Beardall et al. fluorescence in response to Si limitation and re-supply have also been observed in diatoms (Lippemeier et al., 1999). Fv /Fm recovers following re-supply of the limiting nutrient (Geider et al., 1993, La Roche et al., 1993; Young, 1999) and can be used as a basis for bioassays on natural populations of phytoplankton (e.g. Boyd et al., 1996). Following in situ Fe enrichment, Behrenfeld et al. (1996) reported increases in Fv /Fm from ~0.25 to ~0.55 over 24 h which persisted over the 8 days in a HNLC region of the equatorial Pacific. Similar data have been reported following Fe enrichment in other HNLC regions of the equatorial Pacific (Kolber et al., 1994), the sub-arctic Pacific (Boyd et al., 1996, 1998) and most recently, the Southern Ocean (Boyd et al., 1999). Thus measurements of Fv /Fm following re-supply of nutrients are an important index of nutrient status although, if carried out in samples isolated in containers, they could be subject to similar problems to those outlined above for standard bioassays. Nutrient induced fluorescence transients (NIFT) Measurable changes in variable chlorophyll a fluorescence in response to nutrient limitation or re-supply requires several days or hours respectively. Recent observations of more rapid changes in chlorophyll a fluorescence emission, occurring over several minutes, in response to supply of a limiting nutrient offer the possibility of almost instantaneous assessment of phytoplankton nutrient status. These studies have shown that addition of the limiting nutrient to N-limited or P-limited cultures of microalgae will induce characteristic changes in in vivo fluorescence output (Wood and Oliver, 1995; Beardall et al., 1996; Gauthier and Turpin, 1997; Roberts, 1997; Young, 1999; Beardall et al., 2001). The qualitative nature of the response is dependent on the nutrient that is limiting and which nutrient form is re-supplied. Thus NH +4 re-supply to N-limited cells elicits a small rise, followed by a significant drop in fluorescence output, whereas micromolar NO –3 additions to the same cells causes a significant rise in fluorescence which then returns to the initial value over ~10 min (Young, 1999). PO 3– 4 addition to P-limited cells causes a drop of up to 40% in fluorescence emission which recovers to the initial level after several minutes (Beardall et al., 1996). Addition of a nutrient to nutrient replete cells or a non-limiting nutrient generally elicits no response, although Wood and Oliver (1995) reported some non-specific response to NH +4 in P-limited cells of Microcystis aeruginosa. The duration, magnitude and rate of change in fluorescence emission are dependent on the concentration of nutrient added and the nutrient concentrations eliciting half-maximal responses are similar to those for uptake of N and P from the growth medium (Beardall et al., 1996; Roberts, 1997; Young, 1999). Furthermore, in the case of phosphate re-supply, fluorescence transients persist until the external phosphate is depleted, suggesting that the fluorescence transients are associated, at least in part, with uptake processes (Beardall et al., 1996). Observed changes in fluorescence output are mirrored by the well-documented decrease in O2 evolution following re-supply of limiting nutrients (Holmes et al., 1989; Beardall et al., 1996; Gauthier and Turpin, 1997; Young, 1999; Beardall et al., 2001). Turpin and co-workers associated quenching of chlorophyll fluorescence output following NH +4 re-supply to N-limited Scenedesmus minutum (Turpin and Phytoplankton nutrient limitation 61 Weger, 1988; Holmes et al., 1989) with the changes in net C fixation and respiration occurring as a result of N assimilation (see above and Huppe and Turpin, 1994; Gauthier and Turpin, 1997). Clearly any process demanding expenditure of ATP or reducing equivalents derived from the photosynthetic light reactions can influence ATP demand, photosynthetic electron transport and therefore fluorescence output (Figs. 2, 3). Furthermore, work on vascular plant chloroplasts has also shown that elevated cytosolic concentrations of phosphate cause perturbations of photosynthesis and chlorophyll fluorescence quenching resulting from C flux out of the chloroplast to the cytosol (Cerovic et al., 1991) as described above. Wood and Oliver (1995) showed that the nutrient induced fluorescence transient (NIFT) technique can be applied to natural populations, and more recently the responses of phytoplankton populations in Lake Zurich and Lake Lucerne to P resupply were shown to be dependent on the P-status of the two lakes. Only samples from L. Lucerne ([phosphate] < 0.1 µg L –1) showed a NIFT response (Beardall et al., 2001 – this issue). NIFT type analyses clearly show promise as a rapid and sensitive means of detecting limitation of phytoplankton by specific nutrients. Figure 3. A summary of the major processes leading to utilization of ATP and reductant (NADPH, reduced ferredoxin) in an algal cell. C, N and P uptake and assimilation all require ATP while C and N assimilation also require reductant. These demands on intracellular energy pools derived from the light reaction of photosynthesis are competitive. Thus, for instance, the high rates of P uptake that are found in P-limited cells can use up the available ATP to the extent that it is present at insufficient concentrations to support C assimilation. This in turn has a feedback effect on rates of oxygen evolution (see Beardall et al. 2001 – this issue) 62 Beardall et al. However, current work in our, and other, laboratories has yet to thoroughly evaluate the applicability of the technique to natural populations, especially at low cell numbers. Current Challenges and Ongoing Questions Several issues continue to provide methodological problems and challenges for interpreting assays of nutrient limitation in phytoplankton: 1. There are problems extrapolating from laboratory studies to an understanding of nutrient limitation on larger spatial scales. This is illustrated in an extreme case by extrapolations from measurements involving the small volumes associated with flow cytometry to ecosystem or population levels. 2. We need to address nutrient patchiness (see Turpin and Harrison, 1979) and heterogeneity in phytoplankton assemblages, species composition and nutrients within a single sample – flow cytometry offers a way of examining individual species/cells, but can only deal with very limited volumes. 3. How do we extract important data from a mixed sample? How differently do different algal groups respond to nutrient enrichment? How can we define limitation on a population scale if, for example, diatoms are limited by silicate availability, yet other groups are potentially limited by N or P? 4. Changes in species composition can occur with enrichment so that bioassay experiments provide information about limitation of total primary productivity potential and the capacity to increase primary productivity by alleviating different nutrient deficiencies rather than identifying factors limiting growth rates of the initial population. The growth rate of a phytoplankton population may only be marginally limited but enrichment might cause changes in species composition as competition issues come into play. Despite several decades of interest in assessing nutrient limitation in phytoplankton, there remain problems that are common to many of the methods discussed in this review. However each of the methods is characterised by particular advantages and limitations. The best approach is, therefore, not to rely exclusively on one technique but to employ a suite of techniques and parameters to identify nutrient limitation in phytoplankton, which greatly strengthens interpretation of results. The continued refinement of existing methods and identification of useful new indices assessing nutrient limitation of phytoplankton growth requires ongoing input from physiologists to oceanographers and limnologists alike. ACKNOWLEDGEMENTS This contribution was presented as a keynote lecture at the 7th International GAP Workshop, held on 9 –17 Sept. 1999 in Zürich, Switzerland, and supported by the Swiss National Science Foundation (SNF), the Swiss Academies of Natural and Technical Sciences (SANW and SATW), the Swiss Society of Hydrology and Limnology (SGHL), by EAWAG, Zürich Water Supply and the University of Zürich, as well as by Hoffmann-La Roche, Lonza, Novartis, Canberra Packard S.A, Millipore AG and Faust Laborbedarf AG. John Beardall is grateful for the support of the Australian Research Council for his laboratory’s studies on algal physiology. Erica Young acknowl- Phytoplankton nutrient limitation 63 edges support from a Monash Graduate Scholarship, Dr. John Berges for access to an extensive reference collection and Dr Michael McKay for access to unpublished material. Dr. Don Anderson was kind enough to provide early access to papers at proof stage. Note added in proof: Hameed et al. have recently published an interesting comparison of methods for determining nutrient limitation in phytoplankton (Hameed, H.A., S. Kilinc, S. McGowan and B. Moss, 1999. Physiological tests and bioassays: aids or superfluities to the diagnosis of phytoplankton nutrient limitation? A comparative study in the Broads and the Meres of England. Eur. J. Phycol. 34: 253–269. REFERENCES Anderson, M.A. and F.M.M. Morel, 1982. 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