Ethanol Gluconobacter biosensor designed for flow injection analysis: Application in ethanol fermentation off-line monitoring

A microbial amperometric biosensor for the measurement of ethanol in flow injection analysis (FIA) system was constructed. The system used bacteria Gluconobacter oxydans attached on the surface of combined (glassy carbon vs. Ag/AgCl) electrode mounted into the flow cell. The parallel colorimetric glucose determination was used to eliminate non-specific glucose response of microbial biosensor to samples from monitored ethanol fermentation. The current response of microbial biosensor to samples containing both ethanol and glucose consisted of two current portions (ethanol and glucose) whereas the ethanol portion was not influenced by various glucose concentrations. The value of ethanol portion was calculated as the difference of total current signal of microbial biosensor and the portion of signal belonging to glucose obtained from parallel glucose colorimetric determination and glucose calibration curve of microbial biosensor. The response of microbial biosensor to ethanol was linear in the range from 10 μM to 1.5 mM, the response time of the biosensor was up to 3 min. No decrease of response to ethanol was observed during 72 h of continuous measurement with microbial biosensor. Described biosensor FIA system was used for off-line monitoring of ethanol production during alcoholic fermentation. Obtained ethanol concentrations were in a good correlation with a gas chromatography (GC) reference method.