Monitoring of ethanol during fermentation using a microbial biosensor with enhanced selectivity Jan Tkac a,b, * , Igor Vostiar b , Peter Gemeiner a , Ernest Sturdik b a Institute of Chemistry, Slovak Academy of Sciences, Du ´bravska ´ cesta 9, SK-842 38 Bratislava, Slovak Republic b Department of Biochemical Technology, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinske ´ho 9, SK-812 37 Bratislava, Slovak Republic Received 28 June 2001; accepted 6 December 2001 Abstract The present study is concerning the construction of ferricyanide-mediated Gluconobacter oxydans cell ethanol biosensor. The size exclusion effect of a cellulose acetate membrane was used for elimination of glucose interferences during ethanol assays in real samples. A typical response time of the biosensor was 13 s with a high sensitivity of 3.5 AA mM À 1 . The microbial biosensor exhibits a very low detection limit of 0.85 AM and a wide linear range from 2 to 270 AM. The operational stability was excellent. During 8.5 h of repetitive ethanol assays, no decrease in the sensor sensitivity was observed. The biosensor was successfully used in the off-line monitoring of ethanol fermentation with a good agreement with HPLC measurements (R 2 = 0.998). D 2002 Elsevier Science B.V. All rights reserved. Keywords: Amperometric biosensor; Gluconobacter oxydans; Ethanol fermentation; Cellulose acetate membrane 1. Introduction The determination of ethanol is very important for the bioprocess control in fermentation industry. Biosensors are very promising for this purpose, providing necessary infor- mations that allows feedback control. For the construction of ethanol biosensors mainly two enzymes are used. Alcohol oxidase is an expensive and unstable enzyme that is able to oxidise also methanol with oxygen as a co-substrate. NAD- dependent alcohol dehydrogenase needs soluble cofactor (NAD) for active site regeneration and the enzyme kinetics has an unfavourable equilibrium. The application of PQQ-dependent dehydrogenases (PQQ = pyrroloquinoline quinone) is exploited to overcome the disadvantages related to the use of oxidases and NAD- dependent dehydrogenases [1]. These enzymes contain a cofactor (PQQ) that is tightly but not covalently bound to the holoenzyme molecule. Moreover, the oxidation of ethanol by PQQ-dependent alcohol dehydrogenase is irre- versible [2] and the enzyme is unable to oxidise methanol [3]. The main drawback of PQQ-dependent dehydrogenases is the low specific activity and stability after enzyme purification. Microbial biosensors have several advantages over enzyme biosensors: the enzyme does not need to be isolated; the enzymes are usually more stable in their natural cell environ- ment; and the coenzymes and activators are already present in the system [4]. On the other hand, microbial biosensors are less specific compared to the enzyme biosensors. Recently, a method for the analysis of samples contain- ing both ethanol and glucose was developed. This system consists of the non-specific Gluconobacter oxydans sensor, which detects both glucose and ethanol, and the selective glucose sensor which detects only glucose. The concen- tration of ethanol was subtracted from these two signals [5]. A simplified procedure for determination of ethanol selectively in the presence of glucose by mediated G. oxy- dans biosensor using the size exclusion effect of a cellulose acetate membrane is presented here. 1567-5394/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S1567-5394(02)00054-3 * Corresponding author. Department of Biochemical Technology, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinske ´ho 9, SK-812 37 Bratislava, Slovak Republic. Tel.: +42-175-296-7085; fax: +42-125-296-7085. E-mail address: chemtkac@savba.sk (J. Tkac). www.elsevier.com/locate/bioelechem Bioelectrochemistry 56 (2002) 127 – 129