The kinetics of butyric acid fermentation by Clostridium tyrobutyricum at pH 6.0 and 37 degrees C were studied with the wild type ATCC 25755 and its mutant PPTA-Em, which was obtained from integrational mutagenesis to inactivate the chromosomal pta gene, encoding phosphotransacetylase (PTA). The potential of using this mutant to improve butyric acid production from glucose and xylose was evaluated in both free and immobilized cell fermentations. Compared to the wild type, in free cell fermentations PPTA-Em produced 15% more butyrate (0.38 g/g versus 0.33 g/g) from both glucose and xylose, at much higher concentrations (37.2 g/L versus 22.9 g/L from glucose and 33.5 g/L versus 19.4 g/L from xylose). The increased butyrate production in the mutant can be attributed to the reduced acetate production as well as reduced specific growth rate. The acetate yield in the mutant was reduced by 13.5% (0.058 g/g versus 0.067 g/g) and 32% (0.045 g/g versus 0.066 g/g) from glucose and xylose, respectively. The mutant's specific growth rate was reduced by 36% (0.137 h(-1) versus 0.214 h(-1)) on glucose and 26% (0.086 h(-1) versus 0.116 h(-1)) on xylose. A fibrous-bed bioreactor (FBB) was used to immobilize PPTA-Em mutant cells and further improve butyric acid production. The final butyric acid concentrations in fed-batch fermentations reached 49.9 g/L from glucose and 51.6 g/L from xylose, with the butyrate yield increased to 0.44 g/g glucose and 0.45 g/g xylose. As evidenced by the greatly increased butyrate/acetate ratio in the final product profile, it is concluded that the mutant's metabolic pathway has been shifted to favor butyrate production due to the knockout of pta gene even though acetate production remains at a significant level. The observed metabolic shift is corroborated by the changed protein expression patterns as seen in two-dimensional protein electrophoresis and SDS-PAGE. (c) 2005 Elsevier Ltd. All rights reserved.