Lactobacillus buchneri strain NRRL B-30929 was isolated from a fuel ethanol production facility. This heterofermentative, facultative anaerobe can utilize xylose as a sole carbon source and tolerates up to 12% ethanol. Carbohydrate utilization (API, Biomerieux) and Phenotype Microarrays (PM, Biolog) analyses indicated that the strain is able to metabolize a broad spectrum of carbon sources including various monosaccharides (C5 and C6), disaccharides and oligosaccharides, with better rates under anaerobic conditions. In pH-controlled bioreactors, the bacterium consumed xylose and glucose simultaneously at high concentrations (125 g L(-1), pH 6.0). The major fermentation products were lactate (52 g L(-1)), acetate (26 g L(-1)) and ethanol (12 g L(-1)). The strain ferments glucose alone (pH 4.0) into lactate and ethanol with a molar ratio of 1.03:1. This strain will be further explored via genetic engineering for potential applications in biomass conversion.