Propionibacterium acidipropionici, a Gram-positive, anaerobic bacterium, has been the most used species for propionic acid production from sugars. In this Study, the metabolically engineered mutant ACK-Tet, which has its acetate kinase gene knocked out from the chromosome, was immobilized and adapted in a fibrous bed bioreactor (FBB) to increase its acid tolerance and ability to produce propionic acid at a high final concentration in fed-batch fermentation. After about 3 months adaptation in the FBB, the propionic acid concentration in the fermentation broth reached similar to 100 g/L, which was much higher than the highest concentration of similar to 71 g/L previously attained with the wild-type in the FBB. To understand the mechanism and factors contributing to the enhanced acid tolerance, adapted mutant cells were harvested from the FBB and characterized for their morphology, growth inhibition by propionic acid, protein expression profiles as observed in SIDS-PAGE, and H+-ATPase activity, which is related to the proton pumping and cell's ability to control its intracellular pH gradient. The adapted mutant obtained from the FBB showed significantly reduced growth sensitivity to propionic acid inhibition, increased H+-ATPase expression and activity, and significantly elongated rod morphology. Biotechnol. Bioeng. 2009;104: 766-773. (C) 2009 Wiley Periodicals, Inc.