A strictly anaerobic, thermoacidophilic, H-2-producing bacterium was isolated and designated as Thermoanaerobacterium aotearoense. The optimized cultivation conditions for H-2 production are 55 degrees C, pH 6.5 and 10 g l(-1) of glucose or xylose. A metabolic pathway analysis showed that lactate occupied most of the liquid metabolites and consumed a large amount of NADH. To increase the efficiency of hydrogen production, the gene encoding the L-lactate dehydrogenase was knocked out to redirect the NADH flow. Genetic manipulation resulted in the 2 and 2.5 folds increase of the H2 yield and production rate, respectively. The maximum H-2 yields using the Delta ldh mutant were 2.71, 1.45 and 2.28 mol H-2 mol(-1) sugar under glucose, xylose and glucose/xylose mixture tests, respectively. The recombinant Delta ldh strain could ferment the mixture of glucose and xylose to produce H-2 effectively, indicating that the performance of Thermoanaerobacterium in H-2 production can be significantly improved by metabolic engineering technique. (C) 2010 Elsevier Ltd. All rights reserved.