Analytica Chimica Acta 420 (2000) 1–7 Determination of total sugars in lignocellulose hydrolysate by a mediated Gluconobacter oxydans biosensor Ján Tkᡠc a,∗ , Peter Gemeiner b , Juraj Švitel a , Tomáš Benikovský a , Ernest Šturd´ ık a , Vladim´ ır Vala c , Ladislav Petruš b , Eva Hrabárová b a Department of Biotechnology, Faculty of Chemical Technology, Slovak University of Technology, Radlinského 9, SK-812 37 Bratislava, Slovak Republic b Institute of Chemistry, Slovak Academy of Sciences, Dúbravská Cesta 9, SK-84238 Bratislava, Slovak Republic c BIOCEL, a.s., Zahradn´ ı 762, CZ-739 21, Paskov, Czech Republic Received 11 April 2000; received in revised form 11 April 2000; accepted 23 May 2000 Abstract A microbial biosensor for sugar determination was prepared by surface modification of a graphite electrode using Gluconobacter oxydans cells. The sensitivity of amperometric detection was enhanced by using hexacyanoferrate(III) as a mediator. The G. oxydans cells contain membrane-bound aldose dehydrogenase, which catalyses the oxidation of wide range of sugars including all sugars present in lignocellulose hydrolysate. The substrate specificity of the biosensor, effect of pH, temperature, working potential, hexacyanoferrate(III) concentration as well as the physiological state of the cells for de- tection were carefully optimised. The upper value of the linear range of the optimised biosensor was in the range 1.1–2.2 g l −1 for determination of d-glucose, d-galactose, d-xylose, d-mannose and l-arabinose. The biosensor was used for total sugars determination during lignocellulose hydrolysate fermentation. A good correlation between total sugars determined in samples by the biosensor and by quantitative paper chromatography was obtained. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Biosensor; Amperometric; Gluconobacter oxydans; Lignocellulose hydrolysate; Sugar determination 1. Introduction Lignocellulose hydrolysate is the waste from the pulp and paper industry, so the use of hydrolysate as a source of sugars for microbial fermentations im- proves the economy of the process. Lignocellulose is a rich mixture of carbohydrate polymers (cellulose and hemicellulose), lignin, proteins and others compounds [1]. Processing of lignocellulose includes delignifica- tion to liberate cellulose and hemicellulose from their ∗ Corresponding author. Tel.: +421-7-52967085; fax: +421-7-52967085. E-mail address: tkac@chelin.chtf.stuba.sk (J. Tk´ aˇ c). complexes with lignin and subsequent depolymerisa- tion of macromolecular sugars into free sugars. Sul- phite lignocellulose hydrolysate contains a mixture of pentoses and hexoses that can be used as a carbon source for biotechnological production. Actually lig- nocellulose hydrolysate is used mainly for the produc- tion of fuel ethanol using yeasts [2] or recombinant bacterial strains [3] and also for yeast biomass pro- duction [4]. The use of lignocellulose hydrolysate as a substrate is hampered by the presence of inhibitors, such as sulphite, acetic acid and carbohydrate-derived inhibitors, e.g. in birch wood hydrolysate 29 aromatic monomeric compounds, especially aromatic aldehy- des, representing 0.4% of dry weight [5], were deter- 0003-2670/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII:S0003-2670(00)01001-1