Sep 28, 1987 - Curtis A. Suttle2 and Paul J. Harrison. Departments of Botany and Oceanography, University of British Columbia, 6270 University Boulevard.
J. Phycol. 24, 13-16 (1988)
RAPID AMMONIUM UPTAKE BY FRESHWATER PHYTOPLANKTON’ Curtis A. Suttle2 and Paul J . Harrison Departments of Botany and Oceanography, University of British Columbia, 6270 University Boulevard Vancouver, British Columbia V 6 T 1 W5, Canada
patches produced by zooplankton (Lehman and Scavia 1982) lends some credence to the hypothesis. Possibly because of the dogma that oligotrophic freshwater lakes represent phosphorus-limited systems, little effort has gone into discerning whether or not freshwater phytoplankton also possess the transient rapid-uptake characteristic of many marine species. Indications that nitrogen limitation may be more common than previously thought in oligotrophic freshwater systems (Goldman 1960, Lane and Goldman 1984, Priscu and Priscu 1984, Priscu et al. 1985) make this an ecologically as well as a physiologically relevant question. Much of the data on which arguments for the importance of nutrient ‘patchiness’ to phytoplankton ecology have been based were obtained from continuous cultures where by definition ‘patchiness’ does not exist (Rhee 1980). This paper examines ‘surge’ uptake in freshwater phytoplankton grown under non-steady-state, nitrogen-limited conditions.
ABSTRACT
A natural assemblage of freshwater phjtoplankton was removed from a n oligotrophzc lake and grown at N:P supplj ratios of 5:1, 15:l and 45:l (by atoms) i n semicontinuous culture. After a rninimuin of 31 days at a n auerage dazlj growth rate of 0.5 d-I, experiments were conducted examining the time-course of saturated amtnoniuin uptake rates. Culturesgrown under the two lowest LV:P supplj ratios demonstrated greatlj elevated initial, spec@ uptake rates for ammonium and were able to sequester between 7 and 21% of their dailj N requirement i n less than 5 min. The initial rates declined rapidlj but uerefo1lowed by a subsequent increase and decrease over a 120 min period. Key index words: ammonium kinetics; ammonium uptake; N:P supplj ratio; nitrogen limitation; non-steadystate culture; nutrient supplj ratio; uptake transients T h e observation that nitrogen-limited phytoplankton display a several-fold increase in uptake rate for ammonium when exposed to a saturating concentration of the nutrient was first reported by Syrett (1953) for the freshwater chlorophyte Chlorella vulgaris. Fitzgerald (1968) noted that these rates were not sustainable and decreased quite rapidly once the nitrogen deficit was overcome. Conway et al. (1976) documented the response in considerably more detail in marine diatoms and recognized that the enhanced uptake rates consisted of two phases, a short-lived period of very high uptake which was termed ‘surge uptake’ and a longer, sustainable phase which was characterized as ‘internally’ controlled. In a subsequent paper, Conway and Harrison (1977) were able to demonstrate that the magnitude of these responses was species specific, and suggested that these differences might be important in dictating competitive advantage in oligotrophic areas of the ocean. McCarthy and Goldman (1979) emphasized that these highly elevated transients could be a mechanism whereby phytoplankton in nutrient depleted areas sequester nitrogen that was distributed in ephemeral micropatches, and although the existence of such patches has been criticized on theoretical grounds uackson 1980), empirical data showing that phytoplankton can utilize phosphorus
MATERIALS A N D METHODS
A 20 L water sample containing a natural assemblage of phytoplankton was removed from Kennedy Lake on 16 August 1983. This is an oligotrophic lake (ca. 1 pg ch1.L-’) located on Vancouver Island in southwestern British Columbia (49’14‘ N, 125”06’ W). T h e water was filtered through 120 pm screening to remove large grazers and subsequently transported to the laboratory where it was diluted 1 : l with artificial medium (Guillard and Lorenzen 1972). T h e medium was modified by increasing the bicarbonate and silicate concentrations to 300 pM and 200 pM,respectively, propanand replacing Tris buffer (2-amino-2-hydroxymethyl-l,2 diol), which interferes with ammonium analysis with 500 pM MOPS buffer (morpholine propanesulphonic acid). To prevent polymerization the silicate was added as described in Suttle et al. (1986). Nitrogen was decreased to 20 pM and was supplied as NH,CI; phosphorus was supplied as K,HPO, at a concentration of 2.0 pM. When the phytoplankton were in exponential growth as indicated by measurements of in vivo fluorescence they were used to initiate nine 1.5 L semicontinuous cultures. Each culture was diluted daily t o achieve a specific growth rate of 0.5 d-’ and was subjected t o one of three N:P supply ratios (5:1, 15:1, and 45: 1 ;by atoms). Specific growth rate was calculated as -In/, where /is the fraction of the original volume remaining subsequent t o a dilution. Suttle and Harrison (1986) have shown that this method can be used to realize a variety of nutrient-limited states. Concentrations (pM) of ammonium (NH,) and phosphate (PO,) were varied to maintain similar biomasses and were as follows for each of the three treatments (5:1 , 15: 1 , 45: l), respectively: NH, 10.0, 15.0, 22.5; PO, 2.0, 1.0, 0.5. Cultures were grown under continuous light at a n irradiance of ca. 200 pE.rn-*.s-’. Additional details of culturing methods and apparatus are given in Suttle and Harrison (1986). After the cultures had been diluted for a minimum of 3 1 days and community composition had stabilized, NH, uptake rates for each culture were determined. A stable community was defined
‘Accepted 28 September 1987. *Current address and address for reprint requests: Marine Sciences Research Center, State University of New York, Stony Brook, New York 11794-5000.
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CURTIS A. SUTTLE AND PAUL J. HARRISON
as one in which the abundance of the dominant species and in i ~ z t ~fluorescence o varied less than 10% over 3 days. Samples from the 5:l and 15:l cultures were inoculated with ca. 20 pM NH,CI,
and the nutrient disappearance from solution was followed. As residual NH, (ca. 1 pM) was present in the 45: 1 cultures and the uptake rates were much lower, a smaller (ca. 4 p M ) but still saturating concentration of NH, was added. Subsamples (15 mL) were taken immediately upon nutrient addition and at predetermined intervals for 2 h. They were promptly filtered through Whatman GF/F filters which had been pre-treated by washing with 20,mL of 3.7% HCI (by weight), followed by 200 mL of ammonium- and phosphate-free medium and oven drying at 60" C. Filtered standards confirmed that no measurable exchange of NH, occurred on the filters or filtration apparatus. T h e filtrate was analysed for NH, using the technique of Solorzano (1969) as automated by Stainton et al. (1977). Ammonium concentrations were determined from regression equations relating duplicate standards to absorbance (for the 5: 1 and 15:1 treatments5.0, 10.0, 15.0 and 20.0 p M ; for the 45:l treatment-1.0, 2.0, 3.0 and 5.0 pM); r2 for these equations ranged from 0.997 to 1 .OOO. Duplicate NH, determinations always varied less than 0.9% from the means. Prior to each uptake experiment 50 mL of culture was filtered onto ashed (450' C for 6 h) GF/F glass fiber filters and analysed for particulate N (PN) using a Carlo Erba 1106 elemental analyser. Nitrogen-specific uptake rates (h-') were calculated by dividing the measured uptake rate (pmol. L-' .h-') by the PN concentration of the culture at the beginning of the experiment. This routine approach (e.g. Parslow et al. 1984a, b) was taken for two reasons. First, under nitrogen-limited growth the average cell quota at the beginning of the uptake period should be the minimum permitting an average growth rate of 0.5 d-' over the 24 h period between dilutions. Therefore, when the uptake rate is divided by the initial PN concentration, one can directly compare the magnitudeof the uptake rate relative to that required to maintain an average growth rate of 0.5 d-'. For example, an uptake rate of 0.7 h-' is equivalent to 16.8 d-' (0.7 x 24), which is 33.6 times greater than the steady-state uptake rate required to maintain a growth rate of 0.5 d-'. Secondly, when the uptake rates are standardized to the initial PN, changes in the specific uptake rate should reflect changes in the uptake rate per cell. Regardless, correcting for increases in PN over the course of the uptake experiments will not affect the observed pattern other than to exaggerate the decrease in uptake rate over time. As uptake rates were calculated from the difference in concentration in two samples, errors in uptake rates due to errors in NH, determination should be no more than twice that for concentration, or no more than 1.8% of each mean. Any errors in calculated rates resulting from an inaccurate determination of the time between filtrations should be proportional to the error in the time measurement. Such errors should be largest over the shortest time interval, or with respect to our data over the 4 min interval between the first and second filtrations. In the unlikely event that the error in time measurement was as large as 30 s, the resulting error in uptake rate would only be 12.5%,and could not be responsible for the high initial uptake rates observed. It is important to note that there is no reason to expect that errors in NH, or time determinations were biased. Values should be underestimated as frequently as overestimated, which would tend to obscure rather than reinforce any observed patterns. As a single value for PN was used to calculate the specific uptake rates for each culture, any error in PN determination would not affect the pattern of change in uptake rate with time. RESULTS AND DISCUSSION
After 3 1 days the cultures grown at the two lowest N:P ratios (5: 1 and 15: 1) were dominated by a mixed assemblage of species consisting of two diatoms,
probably Synedra radians and Nitzschia holsatica (E. F. Stoermer, pers. comm.), a green alga Scenedesmus sp., and a chroococcoid blue-green, probably Synechococcus sp. In the 45:l (N:P ratio) cultures, only the chroococcoid blue-green was observed. Evidence that the different N:P ratios resulted in different degrees of N and P limitation, and the reasons that a more complex community structure occurred under the lower N:P ratios, are discussed in a further paper (Suttle and Harrison 1988). Nutrient disappearance traces and calculated uptake rates are depicted in Figure 1. Variability in uptake rates over the first sampling interval, among replicate cultures, probably stems from the error involved in measuring small changes in nutrient concentration over short times. T h e average decrease in NH, during the first sampling interval (4 min) in the 5: 1 and 15:1 cultures was 0.6 1 pM, or about fivefold greater than the standard deviation estimated from six 20 pM standards, indicating that the observed pattern of changes in uptake rate with time are real. As would be expected from previous work on marine species (McCarthy and Goldman 1979, Goldman and Glibert 1982, Dortch et al. 1982) elevated uptake rates are only associated with those cultures grown under nitrogen limitation, or in this case at the two lowest N:P ratios (Fig. 1). Measured uptake rates in these cultures, over the 1-5 min interval following perturbation, were between 25 and 71 times the rate required to maintain their average growth rate over 24 h. Consequently, cells exposed to a patch of nutrient, saturating to uptake, could sequester between 7 and 21% of their daily N requirement in less than 5 min. These rates are of the same order as those reported by others (McCarthy and Goldman 1979; Parslow et al. 1984a, b, 1985a, b) for N-depleted cultures of Thalassiosira pseudonm2a (clone 3H), measured over similar time intervals. Our observation that rapid initial uptake rates of NH, were only associated with low N:P supply ratios suggests that this should also be a feature of N-limited natural freshwater phytoplankton communities, as it is for marine assemblages (Glibert and Goldman 198 1). Priscu and Priscu (1984) and Priscu (1987) added saturating additions of 15NH, to lakewater samples and found that the rate of isotope enrichment was considerably elevated over the first several minutes, relative to subsequent intervals. As they and others (Dugdale and Goering 1967, Price et al. 1985) have pointed out, such experiments can be biased by isotope dilution as the result of I4NH, regeneration, although this problem is more likely to be associated with trace additions of I5NH,. As well, the initial uptake rates observed are measurements of gross uptake. Subsequent declines in this rate can be attributed to efflux of the label due to isotopic equilibration of cellular NH, pools, resulting in the measurement of net uptake. Several au-
RAPID AMMONIUM UPTAKE
thors have argued against the significance of this process (Goldman and Glibert 1982, Wheeler et al. 1982, Priscu and Priscu 1984). T h e results we report confirm that the rapid decline in the rate of isotope accumulation observed by Priscu and Priscu (1984) in Lake Taupo was probably not the result of isotope equilibration but resulted from a decrease in the net rate of accumulation. A significant, and to our knowledge previously unreported finding, is the appearance of a rapid but short-term decrease in uptake rate immediately following surge uptake (Fig. 1). There are several mechanisms which could account for this pattern. T h e simplest explanation is that there was a short lag before the NH, could be processed into amino acids, resulting in a temporary reduction in net uptake. Alternatively, the decrease could be the result of a sudden loss of membrane potential due to influx of cations. Ullrich et al. (1984) found the membrane potential in Lernrza gibba (duckweed) was reduced drastically when N-starved plants were pulsed with NH,. T h e decrease in membrane potential was not accompanied by a concurrent reduction in uptake rate, although this could be attributable to the less frequent sampling regime used by them, or perhaps it is not a feature associated with multicellular plants. T h e reason why this response has not been observed in previous culture studies of rapid time course NH, uptake in marine phytoplankton (Goldman and Glibert 1982, Parslow et al. 1984b, 1985a) is unknown; however, such a pattern may only be a feature of ion-dilute environments. Perhaps the most likely possibility is that the short-term decrease in uptake rate may be apparent only in synchronously dividing populations. Work has shown that the cell cycle of certain marine phytoplankton can be strongly entrained to NH, pulses (Chisholm et al. 1984), especially under continuous irradiance; yet short time-course studies of NH, uptake in cultures grown under such conditions have not been reported. Although not explicitly examined, it is reasonable to expect that the phytoplankton in our study were entrained to the daily NH, additions. If the pattern of NH, uptake observed is a reproducible feature of cultures entrained to NH, pulses, then such cultures may prove useful for studying regulatory aspects of NH, uptake. Following the decrease, there was a transient increase and subsequent reduction, respectively, in uptake rate over the 120 min that nutrient disappearance was followed. T h e uptake rates stabilized at a level about 12-fold greater than required to sustain the average growth rate over 24 h. This rate is probably equivalent to the ‘internally’ controlled phase of uptake recognized by Conway et al. (1976). In theory the pattern of changes in uptake rate with time that we observed could result from the mixed assemblage of plankton that were used for these experiments. This seems unlikely, however, as it would require the uptake rates in one subgroup
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TIME (min) FIG. 1 Ammonium uptake rates (solid symbols) and ammonium concentrations (open symbols) for perturbation experiments, on triplicate phytoplankton cultures grown at N:P supply ratios of 5 : 1, 15:1 and 45: 1 . T h e dashed lines represent the ammonium uptake rate averaged over 24 h that would be required to sustain a daily averaged specific growth rate of 0.5 d-’.
of the assemblage to decline rapidly following NH, addition, while an enhanced capacity for uptake was being developed in a different subgroup. Such a lag before maximum uptake rates occur has been reported for phosphate (Suttle and Harrison 1986), nitrate (e.g. Collos 1980) and silicate (Conway and Harrison 1977) uptake, but has not been reported for ammonium uptake. T h e teleological nature of arguments regarding adaptive significance dictates that they should be pursued cautiously. Any of the transient phenomena that have been recognized as being integral with uptake processes for limiting nutrients, could confer competitive advantage under particular regimes of nutrient patchiness. T h e onus must now be to identify the temporal and spatial scales of such patchiness in aquatic ecosystems. T h e authors a r e grateful for financial assistance from the Natural Engineering and Research Council of Canada, the British Co-
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CURTIS A. SUTTLE AND PAUL J. HARRISON
lumbia Science Council, the Federal Department of Fisheries and Oceans, and the Coastal Marine Scholar Program at the State University of New York at Stony Brook. Special thanks are owed to A. M. Chan for help with experiments, to E. F. Stoermer for diatom identification, and to an anonymous referee. Chisholm, S. W., Vaulot, D. & Olson, R. J. 1984. Cell cycle controls in phytoplankton. Comparative physiology and ecology. I n Edmunds, L., Jr. [Ed.] Cell Cycle Clocks. Marcel Dekker Inc., New York, pp. 365-94. Collos, Y. 1980. Transient situations in nitrate assimilation by marine diatoms. 1. Changes in uptake parameters during nitrogen starvation. Limnol. Oceanogr. 25: 1075-81. Conway, H. L. & Harrison, P. J . 1977. Marine diatoms grown in chemostats under silicate or ammonium limitation. IV. Transient response of Chaetoceros debilis, Skeletonema costatum and Thalassiosira gravida to a single addition of the limiting nutrient. Mar. Biol. (Berl.) 43:33-43. Conway, H. L., Harrison, P. J. & Davis, C. 0. 1976. Marine diatoms grown in chemostats under silicate or ammonium limitation. 11. Transient response of Skeletonemu costaturn to a single addition of the limiting nutrient. Mar. Biol. (Berl.) 35: 187-99. Dortch, Q., Clayton, J. R., Jr., Thorsen, S. S., Bressler, S. L. & Ahmed, S. I. 1982. Response of marine phytoplankton to nitrogen deficiency: decreased nitrate uptake vs enhanced ammonium uptake. Mar. Biol. (Berl.) 70: 13-9. Dugdale, R. C. & Goering, J. J. 1967. Uptake of new and regenerated forms of nitrogen in primary productivity. Limnol. Oceanogr. 12:196-206. Fitzgerald, G. P. 1968. Detection of limiting or surplus nitrogen in algae and aquatic weeds. J . Phycol. 4: 12 1-6. Glibert, P. M. & Goldman, J. C. 1981. Rapid ammonium uptake by marine phytoplankton. M a r . Bid. Left. 2:25-31. Goldman, C. R . 1960. Primary productivity and limiting factors in three lakes of the Alaska Peninsula. Ecol. Monogr. 30:20730. Goldman, J. C. & Glibert, P. M. 1982. Comparative rapid ammonium uptake by four species of marine phytoplankton. Limnol. Ocranogr. 27:814-27. Guillard, R. R. L. & Lorenzen, C. J. 1972. Yellow-green algae with chlorophyllide c. J . Phjcol. 8:lO-4 Jackson, G. A. 1980. Phytoplankton growth and zooplankton grazing in oligotrophic oceans. Naturt (Lond.) 284:439-4 1. Lane, J. L. & Goldman, C. R. 1984. Size-fractionation of natural phytoplankton communities in nutrient bioassay studies. Hydrobiologia 118:219-23. Lehman, J. T. & Scavia, D. 1982. Microscale patchiness of nutrients in plankton communities. Science (Wash. D.C.) 216: 729-30. McCarthy, .I. .I. & Goldman, .I.C. 1979. Nitrogenous nutrition of marine phytoplankton-in nutrient-deplet;d waters. Science (Wash. D.C.) 203:670-2. Parslow, J. S., Harrison, P. J. & Thompson, P. A. 1984a. De-
velopment of rapid ammonium uptake during starvation of batch and chemostat cultures of the marine diatom Thalassiosira pseudonana. Mar. B i d . (Berl.) 83:43-50. I984b. Saturated uptake kinetics: transient response of the marine diatom Thalassiosira pseudonana to ammonium, nitrate, silicate or phosphate starvation. Mar. Biol. (Berl.) 83: 51-9. -1985a. Interpreting rapid changes in uptake kinetics in the marine diatom Thalassiosira pseudonana (Hustedt).]. Exp. Mar. Biol. Ecol. 91:53-64. 1985b. Ammonium uptake by phytoplankton cells on a filter: a new high-resolution technique. Mar. Ecol. Prog. Ser. 25: 121-9. Price, N. M., Cochlan, W. P. &Harrison, P. J. 1985. Time course of uptake of inorganic and organic nitrogen by phytoplankton in the Strait of Georgia: comparison of frontal and stratified communities. Mar. Ecol. Prog. Ser. 27:39-53. Priscu, J. C. 1987. Time-course of inorganic nitrogen uptake and incorporation by natural populations of freshwater phytoplankton. Freshwater Biol. 17:325-30. Priscu, J. C., Axler, R. P. & Goldman, C. R. 1985. Nitrogen metabolism of the shallow and deep-water phytoplankton in a subalpine lake. Oikos 45:137-47. Prism, J. C. & Priscu, L. R. 1984. Inorganic nitrogen uptake in oligotrophic Lake Taupo, New Zealand. Can. J . Fzsh. Aquat. Scz. 41:1436-45. Rhee, G.-Y. 1980. Continuous culture in phytoplankton ecology. Adz'. Aquat. Microbiol. 2:151-203. Solorzano, L. 1969. Determination of ammonium in natural waters by the phenylhypochlorite method. Limnol. Oceanogr. 29:799-801. Stainton, M. P., Capel, M. J. & Armstrong, F. A. J. 1977. The chemical analysis of freshwater, 2nd ed. Can. Fish. Mar. Sew. Spec. Publ. 25, 180 pp. Suttle, C. A. & Harrison, P. J. 1986. Phosphate uptake rates of phytoplankton assemblages grown at different dilution rates in semicontinuous culture. Can. J . Fish. Aquat. Sci. 43:147481. -1988. Ammonium and phosphate uptake rates, N:P supply ratios and evidence for N and P limitation in some oligotrophic lakes. Limizol. Oceaizogr. In press. Suttle, C. A., Price, N. M., Harrison, P. J. & Thompson, P. A. 1986. Polymerization of silica in acidic solutions: a note of caution to phyco1ogists.J. Phycol. 22:234-7. Syrett, P. J. 1953. The assimilation of ammonia by nitrogenstarved cells of Chlorella vulgaris. Part I. The correlation of assimilation with respiration. Ann. Bot. 17:1-19. Ullrich, W. R., Larsson, M., Larsson, C-M., Lesch, S. & Novacky, A. 1984. Ammonium uptake in Lemna gibba G1, related membrane potential changes, and inhibition of anion uptake. Phjsiol. Plant. 61:369-76. Wheeler, P. A., Glibert, P. M. & McCarthy, J. J. 1982. Ammonium uptake and incorporation by Chesapeake Bay phytoplankton: Short term uptake kinetics. Limnol. Oceanogr. 27: 1 1 13-28.
