The metabolism of butanol producing bacteria Clostridium acetobutylicum was studied in chemostat with glucose limited conditions, butanol stimulus, and as a reference cultivation. COnstraint-Based Reconstruction and Analysis (COBRA) was applied using additional constraints from C-13 Metabolic Flux Analysis (C-13-MFA) and experimental measurement results. A model consisting of 451 metabolites and 604 reactions was utilized in flux balance analysis (FBA). The stringency of the flux spaces considering different optimization objectives, i.e. growth rate maximization, ATP maintenance, and NADH/NADPH formation, for flux variance analysis (FVA) was studied in the different modelled conditions. Also a previously uncharacterized exopolysaccharide (EPS) produced by C. acetobutylicum was characterized on monosaccharide level. The major monosaccharide components of the EPS were 40 n-% rhamnose, 34 n-% glucose, 13 n-% mannose, 10 n-% galactose, and 2 n-% arabinose. The EPS was studied to have butanol adsorbing property, 70 (butanol) mg (EPS) g(-1) at 37 degrees C. (C) 2016 Elsevier Ltd. All rights reserved.