Effects of N:P atomic ratios and nitrate limitation on algal growth, cell composition, and nitrate uptake’

Scenedesmus sp. was grown in chemostats at a fixed growth rate (p) in an inorganic medium with nitrogen to phosphorus atomic ratios (N:P) ranging from 5 to 80, to investigate the effect of double nutrient limitation. There was no additive or multiplicative effect of the two nutrient limitations: below the optimal cell N:P of30, growth was determined solely by N limitation and above 30, by P limitation. Cell N remained constant up to the optimal ratio and increased linearly with N:P above it. The level of cell P was high at low N:P (N-limited state) but decreased rapidly until N:P approached the optimal and remained constant at a low level at high N:P (P-limited state). Protein was the major fraction in which excess N accumulated under P limitation. Cell free amino acids were a constant proportion of cell N at all N:P ratios. RNA concentration was the same regardless of N:P, it9 level being determined by p independent of the type of limiting nutrients. Cell carbon (C) concentration was higher in the P-limited than in the N-limited state. The C fixation rate per unit chlorophyll a, however, was constant under both Pand N-limited states because the variation in chlorophyll a content was similar to that of C. The apparent maximum uptake rate for nitrate (V) in Nand P-limited cultures decreased with increasing cell N or N:P. In N-limited cultures the half-saturation constant (Km) also decreased at higher cell N or N:P. The variation of V appeared to be affected by the level of free amino acids. Transitions between states of nitrogen contain the two nutrients in an N to P and phosphorus limitation of phytoplankatomic ratio (N:P) of about 15 (Redfield ton growth are common in both lakes 1958; Ryther and Dunstan 1971), I found and coastal seawaters. Such transitions the optimal cellular N:P of Scenedesmus occur seasonally in eutrophic lakes and may be common in estuarine waters where seawater, which is generally N limited but P sufficient, mixes with freshwater having relatively high N and low P levels. Discharge of wastewaters into a body of natural water may also initiate a transition. Information on the effects of such a change in nutrient limitation is scarce. Earlier work (Rhee 1974), however, indicated its importance in determining the composition of a phytoplankton community by influencing competition and succession. Although natural phytoplankton l This work was supported by National Science Foundation grant DEB75-195 19 and Environmental Protection Agency grant R-804689-01. The contents of this paper were presented at an international symposium on the use of algal cultures in limnology (Sandefiord, Norway, 26-28 October 1976). V. Bierman, Jr., and D. Dolan tested the growth models and B. Kusel provided technical assistance. LIMNOLOGY AND OCEANOGRAPHY sp. to be 30; the ratio may vary from species to species. Baule (1918), Verduin (1964), and Droop (1973) suggested that growth during such transitions was controlled in a multiplicative manner. Later, however, Droop (1974) showed in his experiments on phosphate and vitamin B,, limitations with Monochrysis lutheri that growth did not follow a multiplicative pattern but was regulated by the single nutrient in shorter supply. In this study, therefore, I have investigated growth during the transition between the Nand P-limited states. Emphasis was placed on changes in cellular composition because the growth rate (p) of phytoplankton limited by various nutrients is a direct function of the cellular levels of the nutrients (Caperon 1968; Droop 1968; Fuhs 1969; Davis 1970; Rhee 1973; Paasche 1973). Since two nutrient-limited states were involved in this study, the kinetics of N-limited growth and N uptake were also investi10 JANUARY 1978, V. 23(l) Dual nutrient limitation 11 gated to provide baseline information to be used with previous findings on P-limited growth and P uptake (Rhee 1973, 1974). Materials and methods Organism and culture medium-An axenic culture of Scenedesmus sp. was grown in a defined inorganic medium as reported by Rhee (1973, 1974). Nitrate and orthophosphate were thesole sources of N and P. For growth studies at various N:P, the phosphate concentration was kept constant at 3 /.LM while the nitrate levels were adjusted between 15 and 240 PM. For the investigation of Nlimited growth, nitrate and phosphate concentrations were reduced to 21 and 10 PM. Cells were counted in a hemacytometer. For the double nutrient experiments cell volume was calculated by the geometric formula relating volume to cell shape. N-limited growth-Four identical chemostats were made from l-liter (working capacity) Bellco spinner flasks. Each chemostat had a water jacket through which constant temperature water was circulated at 20” + 1°C by a Neslab cooler-circulator. Cultures were stirred with a magnetic impeller unit on a Bellco nonheat-generating magnetic stirrer. Air was passed first through a saturated zinc chloride solution to eliminate traces of ammonia, then through distilled water to saturate it with moisture, and finally through a series of bottles filled with sterile glass wool. Cultures were aerated at about 1 liter min-l. Continuous illumination of about 0.082 ly min-l was provided by white fluorescent lamps coated with Uvinul D-49 (see Rhee 1974). Then cultures were grown at various dilution rates. When a steady state was reached, cells were harvested; the cells and supernatant were stored at -20°C until analysis. Growth at various N:P-Four Bio-Flo chemostats (model C30), each with a working volume of 600 ml, were used. Continuous illumination at about 0.085 ly min-l was provided. The alga was grown at a constant dilution rate of 0.59 d-l, or relative growth rate of 0.437 (p/h:, where h is the maximum growth rate). The variation in dilution rate was ~4%. Aeration was as for the N-limited culture, except that the rate was about 500 ml min-r. Nitrate uptake experiments-The N uptake study was carried out in a manner similar to the P uptake experiments (Rhee 1973). Aliquots (50 ml) of the steady state culture from N-limited chemostats were delivered into a series of 125-ml Erlenmeyer flasks that contained various concentrations of nitrate. The flasks were shaken on a rotary shaker under the same illumination as for N-limited growth studies. Uptake was measured at 20-min intervals by filtering 10 ml of suspension through a membrane filter (Millipore, pore size 0.45 pm) that had been washed with 30 ml of doubledistilled water and determining the amount of nitrate left in the filtrate. Data were analyzed by a nonlinear regression program (Rhee 1973). The rate of N uptake under P limitation was calculated from the 1973 data according to V = (A B)D x cell No.-l, 0) where V is the apparent maximum uptake velocity; A, the nitrate provided in the inflow medium; 23, the residual nitrate level at a steady state; and D, the dilution rate. This study had been done under a 12:12 light-dark cycle, with the light at 0.084 ly min-l. Carbon (C) uptake was calculated from cell C concentration by the equation