Continuous Culture of Rhodotorula rubra : Kinetics of Phosphate-Arsenate Uptake, Inhibition, and Phosphate-Limited Growth

Abstract

The pink yeast Rhodotorula rubra of marine origin was found to be capable of extended growth at very low phosphate concentrations (K,.6 = 10.8 nM). Average intracellular phosphate concentrations, based on isotope exchange techniques, were 15 to 200 mm, giving concentration gradients across the cell envelope of about 106. Sensitivity to metabolic inhibitors occurred at micromolar concentrations. Inability of the phosphate transport system, K. = 0.5 to 2.8 AM, Vmax = 55 umoles per g of cells per min, to discriminate against arsenate transport led to arsenate toxicity at 1 to 10 nm, whereas environmental arsenate levels are reportedly much higher. Phosphate competitively prevented arsenate toxicity. The K1 for phosphate inhibition of arsenate uptake was 0.7 to 1.21LM. Phosphate uptake experiments showed that maximal growth rates could be achieved with approximately 4% of the total phosphate-arsenate transport system. Organisms adapted to a range both of concentration of NaCl and of pH. Maximal affinity for phosphate occurred at pH 4 and at low concentrations of NaCl; however, Vmax for phosphate transport was little affected. Maximal specific growth rates on minimal medium were consistent in batch culture but gradually increased to the much higher rates found with yeast extract media when the population was subjected to long-term continuous culture with gradually increasing dilution rates. Phosphate initial uptake rates that were in agreement with the steady-state flux in continuous culture were obtained by using organisms and medium directly from continuous iculture. This procedure resulted in rates about 500 times greater than one ,p which harvested batchgrown cells were used. Discrepancies between values found and those reported in the literature for other organisms were even larger. Growth could not be sustained below a threshold phosphate concentration of 3.4 nm. Such thresholds are explained in terms of a system where growth rate is set by intracellular nutrient concentrations. Threshold concentrations occur in response to nutrient sinks not related to growth, such as efflux and endogenous metabolism. Equations are presented for evaluation of growth rate-limiting substrate concentrations in the presence of background substrate and for evaluating low inhibitor concentration inhibition mechanisms by substrate prevention of inhibitor flux. Although phosphate is a major nutrient and We first became interested in phosphatekinetic studies of nutrient-limited aquatic mi-limited systems after noting that dilute concrobial systems are fairly common, few data tinuous culture was easier to start after adding exist specifying limiting phosphate concentra-large quantities of phosphate, and that phostions at steady state. This is largely due to the phate seemed to prevent copper sensitivity in fact that phosphate is a usual contaminant of media low in trace metals (11), a phenomenon chemicals and glassware, so that experimental not predicted by usual binding constants (5). systems with low and accurately known phos-Phosphate...

Ammonium uptake and incorporation by Chesapeake Bay phytoplankton: Short term uptake kinetics1

“…Rapid pool exchange has not been examined previously in marine phytoplankton to our knowledge, but has been documented for POd3-in a marine yeast, Rhodotorula rubra (Button et al 1973). At less than micromolar external concentrations, this organism accumulates large internal pools of phosphate, 40% of which exchanges rapidly with external POd3in l-min pulse-chase experiments.…”

“…At less than micromolar external concentrations, this organism accumulates large internal pools of phosphate, 40% of which exchanges rapidly with external POd3in l-min pulse-chase experiments. Significantly, this yeast has a very high capacity PO4 3-transport system, but at high concentrations (~1 PM) the duration of incubation must be decreased to 1 min to avoid cumulative problems of inhibition and product accumulation (Button et al 1973).…”

Multiple-carbon-source-limited growth kinetics of a marine coryneform bacterium

“…Reducing the glucosearginine-based growth velocities by 50% to account for the contribution of substrate supplied by arginine gave equivalent Ka values that were dependent on glucose accumulation rather than on growth rate. At low growth rates, the threshold glucose concentration, A, defined as the minimum value of extracellular glucose, was disproportionately reduced (62%) (5).…”

Kinetic characterization of the two phosphate uptake systems in the fungus Neurospora crassa

“…There are conflicting reports regarding the number and the kinetic properties of the uptake systems of the yeast Saccharomyces cerevisiae (2,3,8,15). The differences may be due, in part, to differences in the culture conditions before assay, since, in studies with other fungi, phosphate uptake ability has been found to be increased after growth in low-phosphate media (4,27,28). The most extensive study of phosphate uptake by a filamentous fungus has been carried out recently with Neurospora crassa (18)(19)(20).…”