Nova Oceanographic Laboratory, Fort Lauderdale, Florida ABSTRACT

Transcription

1 THE PHYSOLOGCAL STATE WTH RESPECT TO NTROGEN OF PHYTOPLANKTO:N FROM LmOsW- NUTRENT SUBTROPCAL WATER AS MEASURED BY THE EFFECT OF AMMONUM ON ON DARK CARBON DOXDE FXA.TONl an Morri~,~ Clarke M. Yentsch, and Charles S. Yentsch Nova Oceanographic Laboratory, Fort Lauderdale, Florida ABSTRACT The ammonium ion enhancement method has been used to estimate the state of nitrogen nutrition in natural populations of marine organisms, with the advantage that the native population can be assessed at time zero. The ammonium ion does not stimulate dark CO, fixation by natural populations of phytoplankton in the low nitrogen subtropical waters of the Florida Strait, indicating that the cells are not physiologically nitrogen deficient; however nitrogen may still bc limiting population size. When enhancement was not noted at time zero, incubation of the water in a carboy resulted in depletion of the available nitrogen and a physiological state of nitrogen deficiency subsequently became measurable by this method. The eventual positive results from the test therefore confirm the negative results at time zero. NTRODUCTON The concept that certain nutrients may limit the growth of natural populations of phytoplankton stems from many early observations of a decline of inorganic phosphorus and nitrogen compounds associated with the growth of phytoplankton. This concept has assumed an important role in the formulation of seasonal models. Changes in cell numbers can be attributed to causes other than a reduction or stimulation of growth; the emphasis in recent years has been to develop methods of detecting whether nutrients are limiting growth. Some methods have emphasized specific nutrients, mostly nitrogen (Eppley et al. 1969; Ryther and Guillard 1959; Fitzgerald 1969). Other methods, directed toward indicating whether a deficiency exists, have been the ratio of carotenoid to chlorophyll pigment (Yentsch and Vaccaro 1958; Manny 1969) and assimilation coefficients (Thomas 1970). l This work was supported in part by the following grants and contracts: NSF GB 7088 and GB 8249 and AFC AT( 40-1) Present address: Department of Botany and Microbiology, University College of London, Gower Street, London WC1 England. 3 Present address: University of Massachusetts, Marine Station, Box 128 Lanesville Station, Gloucester Syrett (1962) observed that CO2 assimilation in darkness was increased when ammonium ion was added to nitrogen deficient cultures. We have confirmed his observations, and our studies with three cultures of marine algae show that there is no increase in dark fixation in nonnitrogen deficient cultures after the addition of ammonium ion, and that this addition has no effect on the capacity of light fixation of C. n this paper we report on the rate of dark fixation after the addition of ammonium ion to cultures of marine phytoplankton and to natural populations of the tropical ocean waters in the Strait of Florida. The response by populations with and without the addition of ammonium ion is a test of the prescncc or abscncc of nitrogen dcficicncy. We are most grateful for the competent technical assistance provided by L. R. Strube, J. C. Laird, and W.?. Owen, Growth METHODS of algae cultures Cultures of the marine algae Dunaliella tertiolecta, Phaeodactylum tricornutum, and LMNOLOGY AND OCEANOGRAPHY 859 NOVEMBER 1971, V. 16(6)

2 860 AN MORRS, CLARCE M. YENTSCH, AND CHARLES S. YENTSCH TABLE 1. Ammonium ion enhancement of dark COz fixation by cultures of Dunaliella tertiolecta and Skeletonema costatilm. At time zero exponentially growing cultures of both algae were transferred to nitrogen-free medium (full growth medium minus KNO,3 and NH&l) or filtered seawater as described in the text. After the time interuals shown in the table, the rate of dark CO, fixation was measured in lhe presence or absence of 0.2,UM NH&l. Resuits expressed as enhancement ratios ~ Time in Dunaliella Skeletonema deficient medium N-free Filtered N-free Filtered (hr) medium sfxwater medium seawater _ Skeletonema costatum were grown in F1 medium ( Guillard and Ryther 1962) at 20C under 20,000 lux fluorescent. light illumination on alternating 12-hr light/12-hr dark cycles for Dunaliella and Phaeodactylum and 14-hr light/lo-hr dark cycles for Skeletonema. Dunaliella cells were harvested by centrifugation after 3 days and Skeletonema cells after 5. Nitrogen deficiency was induced by washing the cells once, resuspending them in nitrogen-free medium (the full growth medium minus Nl&Cl and KN03) or in filtered seawater, and returning them to the growth conditions for various periods. Nitrogen deficient cultures of Phaeodactylum were prepared differently. The alga was grown in 5 liters of modified F1 medium containing no NHdC,l and only 10% of the full-strength concentration of KNO+ This provoked nitrogen deficiency within 4 days. The culture was aerated and cquippcd with a sampling siphon to permit convenient and aseptic sampling. LMeasurement of CO, fixation by algal cultures n experiments with Dunaliella and Skeletonema, 5 ml of the nitrogen deficient cultures were incubated with 0.5 ml of the 14C-NaHC03 ( in a sealed test tube. The concentration of ammonium ion when added was 0.2 ~mole/lit-er and incorporation of radioactivity was measured over 3 hr. n experiments with Phaeodactylum between 1 and 10 ml of the culture were diluted with filtered seawater to 150 ml in the reagent bottles and 1.0 ml of *4C-NaHC0,7 (10 &i/ml) added. The concentration of ammonium ion when added was 6.7 pmoles/liter. n all experiments with algal cultures, the organism was incubated with the radioactive bicarbonate at 20C. Measurement of CO2 fixation by natural phytoplankton populations Seawater was incubated with 1.0 ml of C-NaHC0,7 (10 &i/ml) in 150-ml reagent bottles. The concentration of NH4Cl added where indicated was 6.7 pmoles/ liter. The bottles were incubated in situ, cooled with running seawater on deck where the temperature of the water was 3OC, or incubated at 20C under 20,000 lux fluorescent light, depending on the particular experiment. n some experiments, the phytoplankton was concentrated by passage of seawater through a No. 20-mesh plankton net or by filtration through a glass-fiber disk in a submersible batch filtering unit (Laird et al. 1967). The filters wcrc cut in equal portions and placed in sterile petri plates with filtered seawater and 0.5 ml of 14C- NaHCj03 ( 10 &i/ml), and 1 ml of ammonium chloride as indicated. After 4 hr in an incubator on deck, the pieces of disk were transferred to 10 ml of 5% acetic acid in ethanol, dried, and radioactivity on them was counted using a Picker compact scaler. The routine experimental procedure in all other experiments was incubation of four dark bottles, two with ammonium ion added and two without. Bottles were darkened by covering with metal foil or black electrical tape. Although not necessary to this technique, two light bottles, with and without ammonium ion, were run with each experiment as a check for the amount of light fixation of CO2. After incubation with the j4c-nahc03, the plankton suspension was Eiltercd through A 0.45-p 25-mm Milliporc filters. The filters were dried and the radioactivity counted.

3 DARK CO2 FXATON AND PYTOPLANKTON NTROGEN O * TME EXPERMENTS PERFORMED N DAYS FG. 1. Carbon-14 sodium bicarbonate incorporated in darkness by a diluted Phaeoclactylum culture with ( +) and without (-) the addition of ammonium ion. Upper portion is enhancement ratios (D+: D-). Samples for experimental purposes were taken from the main culture daily,

4 862 AN MORRS, CLARCE M. YENTSCH, AND CHARLES S. YENTSCH l i 5 Y Phaeodactylum culture-fig Coastal water carboy-lauderdale Marina, 19 Jan 1970-Fig Open ocean carboy- Dee 1969-Fig RESULTS Ammonium ion enhancement in cultures n healthy growing cultures of DunuZieZZa and Skeletonema, the addition of ammonium ion to the medium does not influence the rate of dark fixation (Table 1). Yet when the same cultures are transferred to media where ammonium ion, nitrate, or nitrite are absent, the enhancement ratio ( dark fixation with added ammonium ion: dark fixation with no added ammonium ion) increases from 1.2 to 14.3 in 72 hr. Enhancement of dark fixation is also observed when cultures are filtered and resuspended in waters collected from the surface of the Strait of Florida. This demonstrates that the levels of ammonium- N, nitrate-n, and nitrite-n are very low in these waters. The effect on the rate of dark fixation of adding ammonium ion to a nitrogen deficient culture of Phaeodactylum is shown graphically in Fig. 1. The experiment was prepared as follows. The diatom was grown in a modified F1 medium, as described above, under 20,000 lux at 20C. Samples were taken for the measurement of chlorophyll and the rate of light and dark fixation of 14C, n the case of the dark fixation samples, half receive added ammonium ion (see methods), The culture grew for about 4 days ( Table 2) and enhanced dark fixation after the addition of ammonium ion occurred after 2 days ( Fig. 1). A maximum at 5 days was followed by a decline. A second maximum appeared after 16 days, about half as large as the first. Figure 1 shows that the onset of enhancement is abrupt, that is, the slope (enhancement/time) to the maximum is steep. The small changes in the rate of dark fixation without the addition of ammonium ion is probably due to changes in cell density that occurred during the experiment. n summary, we can say that the dark fixation mechanism in marine phytoplankton responds as in Chlorella (see Syrett 1962 ). When nitrogen is limiting, the addition of ammonium ion greatly stimulates the rate of dark fixation. n dense cultures we observed enhancement ratios greater than 10. Our data are meager, but apparently the signaling of nitrogen limitation by ammonium ion enhancement of dark fixation occurs out of phase with cell divi- sion; that is, high enhancement ratios occur before cell division ceases entirely. Enhancement as a function of population size Elsewhere ( Morris et al. 1971) we have described the rate of dark fixation in relation to the number of cells present. As implausible as it sounds, the highest rates of dark fixation per cell occur in sparse populations of the open oceans and the lowest in dense populations or cultures. Therefore there are at least two possible means

5 DARK CO2 FXATON AND PHYTOPLANKTON NTROGEN O ML DLUTED ML DLUTED+150 ML DLUTED A t i FG 2. Carbon-14 sodium bicarbonate incorporated in darkness by various dilutions of a nutrient deficient (4 day) culture of HmeoductyZum with ( +) and without (-) the addition of ammonium ion. Upper portion is enhancement ratios ( D+ : D-). of externally regulating dark fixation: one by the addition of ammonium ion; the other, by the number of cells present. Conceivably there can be an upper limit for the rate of dark fixation per cell that could be reached or approached by having few cells in suspension. f this is the case, and a nitrogen deficiency is present, the addition of ammonium ion would have little influence on the rate of dark fixation. To test this idea, we have studied ammonium ion enhancement of dark fixation using three concentrations of algae. Cultures were prepared as described above. The three concentrations resulted from dilution of a culture of Phaeoductylum with filtered, nutrient impoverished, surface ocean water. The three concentrations ( Fig. 2) can be thought to represent the extremes of culture conditions with high cell numbers to open ocean conditions with low cell numbers. The time course of ammonium ion enhancement of dark fixation is shown for all three. n the case of high cell numbers, (Fig. 2A), the enhancement ratio reached a maximum of 5.0 after 5 hr. n the intermediate culture ( Fig. 2B) with half the cell number elf the previous culture, enhancement reached a maximum of 3.0 after 3 hr. n the culture with the lowest number of cells ( Fig. 2C ), roughly comparable to the open ocean, the culture enriched with ammonium ion exhibited barely significantly higher rates of dark fixation than the unenriched culture, and the enhancement ratio was only slightly greater than 1.0. The experiment shows the effects of both cell density and ammonium ion enhance-

6 864 AN MORRS, CLARCE M. YENTSCH, AND CHARLES S. YENTSCH - 1 /ooo- +, )+ t + \ \ + \p/- \ s. + +\ \+ \ 0. l d TME WHEN EXPERMENT PERFORMED N DAYS FG. 3. Carbon-14 sodium bicarbonate incorporated in darkness by phytoplankton from coastal water with ( + ) and without (-) the addition of ammonium ion. Upper portion is enhancement ratios ( D+: D-). Samples for experimental purposes were taken from the carboy daily.

7 DARK CO2 FXATON AND l?ytoplankton NTHOCEN AUL.E 3. Nutrient data, chlorophyll data, ancl enhancement ratios (D+:D-) for various experiments with natural populations of marine phytoplankton collected from surface waters sta. NO. 1 3 G NOs-N NOs-N (fig atom/liter) OS-P Chl (mg/m31 Carhoy from pollirted coastal water-pig. 3-start of experiment Ocean water carboy-fig. 4-start of experiment Horizontal section of Florida Strait-16 October a Chl values after concentrating Phytoplankton collected through No. 20-mesh netting Oscillatoria erythraea (individually selected after collecting in No. 20 net) Phytoplankton on glass-fiber disk using submersible batch filtration unit D+:D ment on the rate of dark fixation. Although the addition of ammonium ion to nitrogen deficient cultures having large cell numbers greatly stimulates dark fixation, under similar conditions with low cell numbersusing cells of the identical physiological state-the stimulation is much less. Ammonium ion enhancement in natural populations The fact that two factors are responsible for the rate of dark fixation complicates interpretation of the enhancement ratio. To use the ratio quantitatively, that is to estimate the degree of nitrogen deficiency, would depend on knowing the biomass of phytoplankton. This in itself is a difficult problem. n spite of this drawback, ammonium ion enhancement can be used as an all or nothing indicator of nitrogen deficiency in natural populations. n the first experiment, a population was chosen in which the possibility of nitrogen deficiency was remote. Water collected from a coastal estuary was placed in a 12- liter carboy, which was slowly stirred and illuminated with 20,000 lux on a 14-hr light/ O-hr dark cycle at 20C (about the ambient temperature of the surface water in the estuary where the sample was collected). Periodically samples were removed by siphoning. All samples were inoculated with 14C; half were enriched with ammonium ion, the other half were not. Roth groups were incubated in darkness at 20C for 4 hr. The time course of dark fixation for the population is shown in Fig. 3. After about 3 days of little or no enhancement the dark fixation rate increased markedly, with enhancement ratios being in excess of 2.0 on day 4. After this, enhanccmcnt declined slowly. There is a marked similarity between the response of this natural population ( Fig. 3) and the culture experiments (Fig. 1). Our interpretation is that as the population

8 AN MORRS, CLARCE M. YENTSC, AND CARLES S. YENTSC 2.0~ ot, L d/ O / 12 TME WHEN EXPERMENT PERFORMED N DAYS FG. 4. Carbon-14 sodium bicarbonate incorporated in darkness by phytoplankton from ocean water with ( + ) and without (-) the addition of ammonium ion. Upper portion is enhancement ratios (D+: D-). Samples for experimental purposes were taken from the carboy daily. grew, the reservoir of nitrogen in the carboy deficiency. nitially the population was not was depleted and growth arrested. The nitrogen deficient, indicated by the relamaximum enhancement ratio occurring on tively high levels of both nitrate-n and day 4 signaled the onset of the nitrogen phosphorus present in the estuary (Table 3).

9 DARK CO2 FXATON AND PYTOl?LANKTON NTROGEN 867 A second experiment, prepared exactly as the previous one, utilized natural phytoplankton populations taken from the surface waters in the Strait of Florida ( Fig. 4 ). The population size as indicated by chlorophyll concentration (Table 2) is similar to that of the coastal area; however, both nitrate-n and phosphorus levels were considerably lower in the open ocean water (Table 3). n the latter case, the rate of dark fixation almost immediately showed the effects of the added ammonium ion. By the second day, an enhancement ratio of almost 1.8 occurred after which there was a decline. Our interpretation is that this population quickly depleted the avail- able nitrogen, as indicated by the maximum enhancement ratio after 1 day of incubation. This experiment complements the previous experiment conducted in coastal estuarine conditions, that is, nitrogen deficiency would have been predicted to appear earlier because of the lower nitrogen concentration. However, even with the abrupt appearance of the deficiency, there is no indication that the population was deficient at the start of the experiment. n both the coastal and oceanic surface water experiments, the dark uptake without enrichment of ammonium ion also changed during the experiment. We think that this is due to changes in cell numbers during the experiment, but this has yet to be confirmed. Other experiments have been conducted in the ocean. Table 3 shows ratios for enhancement after a 4-hr experiment, Because of the distribution of the density field across the Strait of Florida, water higher in nutrients is nearer the Florida side, One would suspect that if nitrogen deficiency occurs, it would be more pronounced in populations near the center of the strait. Our enhancement experiments do not show this. However, the significance of the measurements is in question because of the small numbers of cells (see Fig, 2). These cells would already have a high rate of dark fixation; the addition of ammonium ion will only slightly increase it, One way to overcome this problem is to concentrate the cells from seawater and resuspend them in a smaller volume. Carboys with plankton concentrates have been used for a time course, n one experiment, a plankton net was used; on another occasion, a submersible batch filtration unit (Laird et al. 1967) was used for concentration of the plankton. n either case, very little and probably insignificant enhancement occurred (Table 3). DSCUSSON A growing body of information suggests that the phytoplankton of the open ocean is not nitrogen deficient, at least to the degree that can be achieved in cultures and that was previously thought to occur in natural populations (see Yentsch 1962). Some workers have suggested gradations of deficiency ( Thomas 1970 ) ; others distinguish between limitation and deficiency. The technique of ammonium ion enhancement suggests that nitrogen deficiency does not exist in tropical oceanic populations to the degree that it can be induced in cultures. This is in agreement with the findings of Thomas ( 1970) and Eppey et al. ( 1969) for Pacific oceanic waters. The question is, why are populations in tropical oceanic waters so small? We believe that the population size in these areas is limited by nitrogen in the same manner that a culture would be limited by the level of nitrogen introduced into a chemostat. Population size is a balance between nutrient input and that part of the population re- moved by grazing etc. The distinction between limitation by nitrogen and nitrogen deficiency is that physiological processes are not markedly affected by the limitation. These ideas are not particularly novel. They support the concept that ammonium ion is rapidly recycled (Dugdale 1967) and that net growth is a function of added nitrogen in the fixed forms. The value needed is the growth rate and the problem will not be solved until that value is obtained. t might be possible to utilize ammonium ion enhancement, calibrated for cell number, to arrive at a growth rate. This would be dependent on the precision of the 14C technique. We believe that it would not bc fruitful. The enhancement phenomenon, however, can bc useful in studies of regeneration. Some of our experiments indicate

10 868 AN MORRS, CLARCE M. YENTSCH, AND CHARLES S. YENTSC that deamination of phytoplankton organic material may have occurred during the latter part of the experiments, Low enhancement ratios generally occurred after the maximum enhancement. The oscillation between maxima and minima may represent a recycling of nitrogen within the system. The observations on dark fixation in the presence or absence of ammonium ion again raise the question of what to do with the dark fixation value when computing productivity. This is not too serious an error in extremely productive areas, but most of the oceans are not like this, and dark fixation values as high as 50% of the carbon fixed in the light are frequent. We do not have enough information on the influence of the ammonium ion in the dark fixation process in the natural environment. owever, because the phytoplankton rapidly assimilates carbon in the presence of ammonium ion in the dark bottle, subtraction of the dark bottle fixation in this case provides carbon fixation estmiates that arc far too low. REFERENCES DUGDALE, R. C Nutrient limitation in the sea: dynamics, identification, and significance. Limnol. Oceanogr. 12 : EPPLEY, R. W., S. L. COATSWORTH, AND L. SOL&- ZANO Studies of nitrate reductase in marine phytoplankton. Limnol. Oceanogr, 14: FTZGERALD, G. P Field and laboratory evaluations of bioassays for nitrogen and phosphorus with algae and aquatic weeds. Limnol. Oceanogr. 14: GULLARD, R. R. L., AND J. H. RYTHER. 1962, Studies on marine planktonic diatoms 1. Can. J. Microbial. 8: LARD, J. C., D. P. JONES, AND C. S. YENTSCH A submersible batch filtering unit. Deep-Sea Res. 14: MANNY, B. A The relationship between organic nitrogen and the carotenoid to chlorophyll a ratio in five freshwater phytoplankton species. Limnol. Oceanogr. 14: MORRS,., C. M. YENTSCH, AND C. S. YENTSCH Relationship between light carbon dioxide fixation and dark carbon dioxide fixation by marine algae. Limnol. Oceanogr. 16: RYTEER, J. H., AND R. R. L. GULLARD Enrichment experiments as a means of studying nutrients limiting to phytoplankton production, Deep-Sea Res. 6: SYUXTT, P. J Nitrogen assimilation, p n R. A. Lewin [ed.], Physiology and biochemistry of algae. Academic. THOMAS, W. H On nitrogen deficiency in tropical Pacific oceanic phytoplankton: photosynthetic parameters in poor and rich water. Limnol. Oceanogr. 15 : YENTSCH, C. S Marine plankton, p n R. A. Lewin [ea.], Physiology and biochemistry of algae. Academic. -, AND R, F. VACCARO Phytoplankton nitrogen in the oceans. Limnol. Oceanogr. 3: