A glucose sensor for fermentation monitoring
The evaluation, analysis and development of an oxygen-insensitive amperometric glucose biosensor and its application in microbial batch culture are described. The biosensor consisted of a graphite foil electrode modified with glucose oxidase and 1,1'-dimethylferrocene, and operated via mediated electron transfer from the enzyme to the electrode. Initial evaluations illustrated several operating characteristics which would be expected to cause problems in continuous monitoring applications, most notably sensor instability and a progressive increase in response time. The main underlying causes of these unfavorable characteristics were identified as enzyme loss, mediator loss and substrate diffusion limitation within the electrode. As a consequence of these insights, further development of the sensor was undertaken. A number of different electrode materials and enzyme immobilization techniques were tested, resulting in the development of a novel immobilization procedure using a hexadecylamine coating to bind 'the activated carbohydrate residues of periodate-oxidized glucose oxidase. This improved the sensor lifetime and response time under continuous operation. Strategies for the reliable application of the biosensor in fermentation monitoring were evaluated. In-line flow cell and in_§itu membrane probe approaches were considered, and the latter approach was preferred: Considerable attention was devoted to optimising the design of such probes. The best design accommodated a three electrode configuration with a multiple biosensor array. It was found necessary to allow for periodic on-line calibration within the aseptically operating probe. This configuration was successfully applied on-line to monitor glucose in batch cultures of Escherichia coli.