Abstract: The mechanism and kinetics of the glucose uptake systems of three representative microorganisms are studied during cultivation in a chemostat. The three microorganisms are Lactococcus cremoris, Escherichia coli, and Saccharomyces cerevisiae. Two models describing respectively competitive and independent uptake of the two glucose anomers are tested on experimental data where α- and β-glucose are determined by flow injection analysis after pulse addition of the pure anomers to a chemostat. The very accurate experimental results are used to give a convincingly clear model discrimination for all three microorganisms. The uptake of glucose by S. cerevisiae occurs by a competitive mechanism with preference for α-glucose (Kα = 32 mg/L and K(beta) = 48 mg/L). Surprisingly, the glucose uptake by the two bacteria is shown to be mediated by anomer specific transport systems with no competitive inhibition from the other glucose anomer. This novel finding has not been described in the literature on the phosphotransferase system. In L. cremoris the relative uptake rates of the glucose anomers match the equilibrium composition exactly (36% α- glucose). In E. coli the relative uptake rate of α- glucose at glucose unlimited growth is 26%, which means preference for β-glucose. However, the saturation constants of the two sites in E. coli are Kα = 2 mg/L and K(beta) = 15 mg/L, and a preference for α-glucose is exhibited at very low glucose concentrations. The results are of considerable importance in relation to enzyme based on- line measurements during fermentations as well as to the modeling of glucose limited growth and product formation.

Abstract: Samples (8 ml) of fermentation liquor are mixed with 0.8 mL of 6.6 M HClO4 and cooled in ice for 15 min to quench metabolic activity, then adjusted to pH 6.3 with 6.6 M KOH and diluted to 12 mL. The ppt. is centrifuged off and 500 µL of supernatant solution is injected into 10 mM phosphate buffer of pH 6.3. The stream is passed through a reactor containing lactate oxidase or glucose oxidase immobilized on a nylon tube, and is then merged with luminol - Fe(III) reagent for spectrophotometric determination of the H2O2 produced. A reactor in which the nylon tube was knitted in a figure-of-eight shape around two glass spatulas (diameter 4 mm) gave better conversion than one with the tube coiled around a cylinder (diameter 10 mm). With use of the purer (USB 16135; 300 iu mg-1) of two glucose oxidase preparations, the calibration graph (based on peak heights) was rectilinear up to almost 200 mg l-1. The upper rectilinear limit for lactic acid was ~50 mg l-1. Limits of determination were 50 and 150 µg L-1 for lactic acid and glucose, respectively. The technique was used to study bacterial and yeast fermentations, especially the product formation pattern at very low glucose concentration. and the microbial preference for α- or β-glucose. Flow injection analyzers for glucose and lactate were optimized for the measurement of very low concentrations. in complex fermentation media. The analytes were enzymatically oxidized and the H2O2 formed was detected by chemiluminescence. The enzymes were immobilized on a nylon tube and, by measuring the flow-rate dependence of the conversion of H2O2 by catalase, the performance of differently combined enzyme reactors was evaluated. In the resulting glucose and lactate analyzers, the limits of determination in aqueous solutions were 50 µg lactic acid/L and 150 µg glucose/L. When measuring glucose concentrations. in the range 0.5-10 mg/L, the purity of the enzyme used in the analyzer must be high. The analyzers were used to study bacterial and yeast fermentations, in particular the product formation pattern at very low glucose concentrations. and the microbial preference for α- and β-glucose.