Operation and performance of analytical packed-bed reactors with an immobilised alcohol oxidase

Hansenula polymorpha alcohol oxidase (AOX) was immobilised on propylamino-derivatised controlled pore glass (CPG) by covalent attachment using glutaraldehyde (GA) as cross-linker. Different pore and particle sizes were used as well as different experimental conditions (GA concentration, buffer type, pH, time of activation) to optimise the enzymatic productivity and operational stability of the immobilised enzyme. The best results were obtained by activating CPG with 6.5% GA in phosphate buffer, pH 7, for 1 h. The highest activity (0.45 U/mg) and productivity (6.2 mol/m3 min) were obtained with a CPG support with 120–200 mesh and 550 Å pore size. Mini packed-bed bioreactors (9.4–69 mm3) with the immobilised AOX, were used to monitor ethanol. The performance of the bioreactors was simulated using a plug flow model. External mass transfer limitations were observed for residence times higher than 2 s. The bioreactors operated continuously at 32 °C for more than 14 h without significant loss of performance (less than 5%). Ethanol in real samples such as beer, brandy and fermentation media was also successfully monitored. Bi-enzymatic bioreactors containing AOX and HRP were further constructed and displayed a similar performance.