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.