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 P-and 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 N-and 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.

SUMMARY The kinetics of phosphate uptake and growth in Scenedesmus sp. have been studied in continuous culture with particular reference to the shifts in the cellular P compounds as a function of growth rate. Uptake velocity is a function of both internal and external substrate concentrations and can be described by the kinetics of noncompetitive enzyme inhibition. The concentrations of polyphosphates (alkali‐extractable or 7‐min) can he substituted as inhibitors in the kinetic equation. The apparent half‐saturation constant of uptake. Km, is 0.6 μM. The apparent half‐saturation concentration for growth is less than Km, by 1 order of magnitude. Growth is a function of cellular P concentrations, and the polyphosphates (alkali‐extractable or 7‐min) appear to regulate growth rate directly or indirectly. To understand P limitation, therefore, it is necessary to measure both external P and internal polyphosphate levels. Evidence indicates that alkali‐extractable polyphosphates, which can be quantitatively determined by a simple method of measuring surplus P, are involved in cell division process find that a maintenance concentration of functional phosphate exists in the form of poly phosphates. Alkaline phosphatase activity has an inversely linear relationship to growth rate and to the reciprocals of both polyphosphates and surplus P. Changes in lipid P, RNA P, and presumably all other forms except DNA are related to changes in growth rate.

The combined stress of nutrient limitation and suboptimal temperature on growth was studied with turbidostat and chemostat cultures of Scenedesmus sp. and Asterionella formosa. The combined effects were greater than the sum of individual effects and were not multiplicative.In N-and P-limited Scenedesmus sp. and A. formosa the cell quotas (4) of both limiting and nonlimiting nutrients increased with decreasing temperature. At a given temperature cell quotas of limiting nutrients ,also increased with the growth rate (p) and followed a saturation function. Higher values of the minimum cell quota (9J at lower temperatures show that cells require more nutrient with decreasing temperature.The change of q,, with temperature varies with the type of limiting nutrient. This change for N and P in Scenedesmus sp. suggests that their optimum ratio, the ratio at which one limitation changes over to the other, is higher at suboptimal temperatures.Cell quotas of nutrient-sufficient cultures (qm) for C, N, and P and cellular chlorophyll a concentration increased with decreasing temperature. The quota of each nonlimiting nutrient in nutrient-limited cultures had the same value as 9m. RNA decreased with temperature.

The optimum atomic ratio of N to P, the ratio at which one nutrient limitation changes over to the other, was determined in seven species of freshwater planktonic algae. The ratio varied over a wide range among species; the average for these species was 17. If the cellular nutrient ratios in marine species are comparable with those in freshwater organisms, Redfield's ratio of 15 is remarkably close to the average. Cellular N:P ratios varied over a 24‐h period under a light:dark cycle. The variation of the optimum ratio between species and diel change in cellular N:P ratios within a species could play an important role in population dynamics by enhancing the probability of coexistence of species.

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