In this study, a new bacterial strain MEL09, which produces acetoin at high concentrations, was isolated from solid cultures of traditional Chinese vinegar. Based on physiological and biochemical characteristics as well as the 16S rDNA gene sequence, strain MEL09 was identified as Bacillus licheniformis. To improve acetoin production by B. licheniformis MEL09, medium composition and culture conditions were optimized by varying one factor at a time and using orthogonal array tests. Under these optimized conditions, the maximum acetoin concentration achieved was 41.26 gl(-1), with 41.26% glucose conversion efficiency (84.39% of theoretical glucose conversion efficiency). This increase is 84.86% over the initial condition and is, to our knowledge, the highest acetoin level ever reported using fermentation methods. (C) 2010 Elsevier Ltd. All rights reserved.