Thermoanaerobacterium saccharolyticum is a thermophilic anaerobe that was previously engineered to produce ethanol at high yield from a wide array of biomass-derived sugars. We evaluated  this organism for Simultaneous Saccharification and Fermentation (SSF) of pretreated hardwood. Improvements in inhibitor tolerance, ethanol production to high titers and cellulose conversion were made using directed and evolutionary approaches to be described herein. A draft genome sequence has enabled the use of microarrays to study the effects of ethanol and inhibitors on gene transcription. Combined with GC-MS metabolite analysis, we have identified potential bottlenecks in the ethanol pathway.  Methods were developed that allowed directed genetic manipulations, use of knockout / overexpression libraries, mutagenesis and growth selection to yield strains with increased performance characteristics, producing greater than 60 g/L ethanol in sugar fermentation or SSF.