n-butanol fermentation by Clostridium beijerinckii ATCC 10132 was investigated by optimization of various physiological and nutritional parameters. Nitrogen source and metal ions played a crucial role on the titre of n-butanol produced in the fermentation. Optimization of the parameters resulted in about 6-fold increase and an unprecedented titre of 20 g L-1 of n-butanol in 72 h incubation period. The process could be scaled up to 3 L level with similar titres. The strain was found to be tolerant to 25 g L-1 n-butanol under optimized conditions and the tolerance could also be demonstrated by the strain's ability to accumulate rhodamine 6 G. Adaptation of the strain to increased solvent in the medium was accompanied by increased expression of the chaperone, GroEL and change in fatty acid profile of total lipids. This is the first report wherein high titre of n-butanol has been obtained in single batch fermentation without resorting to solvent stripping or strain improvement. (C) 2011 Elsevier Ltd. All rights reserved.