ABSTRACT
Analysis of continuous culture methodology suggests that this potentially powerful tool for kinetic analysis can be improved by minimizing several inherent shortcomings. Medium background substrates - organic carbon, phosphate, and manganese - were shown to dominate kinetic observations at concentrations below chemical detection methods. Reactor wall growth, culture size distribution changes, sample removal-induced steady state perturbations, and limiting substrate leakage from organisms are treated in terms of kinetic measurement errors. Large variations in maximal growth rates and substrate uptake rates found are attributed to experimental protocol-induced transient states. Relationships are presented for correcting limiting substrate concentrations for lability during sampling, contamination with unreacted medium, and background substrate effects. Analytical procedures are discussed for improved measurement of limiting substrate kinetics involving enzymes, isotopes, and material balance manipulation. Relaxation methods as applied to continuous culture are introduced as a means for isolating separate rate constants describing net substrate transport and for evaluating cellular metabolite leakage. Low velocity growth, multiple substrate metabolism, and endogenous metabolism are discussed along with measurements showing that 1-month generation times for aquatic microorganisms can be quite normal and that the kinetics are compatible withµg/liter limiting substrate concentrations. The concept of regarding growth kinetics as the sum of several net accumulation processes is suggested.
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 10(6). Sensitivity to metabolic inhibitors occurred at micromolar concentrations. Inability of the phosphate transport system, K(s) = 0.5 to 2.8 mum, V(max) = 55 mumoles 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 mum. 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.
