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

Institute of Marine Science, University of Alaska, College, Alaska 99701
Department of Biophysics and Genetics, University of Colorado Medical Center, Denver, Colorado 80220

ABSTRACT

The pink yeast Rhodotorula rubra of marine origin was found to be capable of extended growth at very low phosphate concentrations (K_0.5 = 10.8 nM). Average intracellular phosphate concentrations, based on isotope exchange techniques, were 15 to 200 nM, 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_s = 0.5 to 2.8 µM, V_max = 55 µmoles 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 K_i for phosphate inhibition of arsenate uptake was 0.7 to 1.2 µM. 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, V_max 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 culture. This procedure resulted in rates about 500 times greater than one in which harvested batch-grown 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.

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