Development of a biosensor for monitoring of glycerol during alcoholic fermentation

A biosensor for the measurement of glycerol in FIA was constructed using covalently immobilized glycerokinase and glycerol-3-phosphate oxidase in conjunction with a Pt based hydrogen peroxide probe. Different immobilization strategies have been studied including random and asymmetric immobilization onto a polymeric support and immobilization onto two different membranes. The latter resulted in the best configuration for batch measurement. The most effective configuration for measurement in FIA was the immobilization of glycerokinase in a glass beads reactor coupled with glycerol-3-phosphate oxidase on a preactivated Immobilon AV membrane kept at the electrode surface. Using a 250-microliter injection loop, 3 mmol ATP(Mg+2) in 0.1 M borate buffer pH 8.5 and a flow rate of 0.5 ml/min, a linear response in the 2 x 10(-6)/10(-3) mol/l range and a detection limit of 5 x 10(-7) mol/l were obtained for glycerol. Lifetime of the glycerol-3-phosphate membrane was extended up to 1 month by storage in the working buffer containing 1% DEAE-dextran and 5% lactitol. More than 350 samples can be assayed with this system. The biosensor was used to monitor off-line glycerol production during alcoholic fermentations carried out at different pHs and temperatures.