S115: Development of thermophilic bacteria for the conversion of lignocellulosic biomass to ethanol

Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic anaerobes with efficient and complementary capabilities of converting cellulosic materials to ethanol and organic acids. C. thermocellum is one of the most effective cellulose-utilizing organisms while T. saccharolyticum readily depolymerizes and ferments complex hemicelullosic material. The development of these organisms for the industrial conversion of lignocellulosic biomass to ethanol requires metabolic engineering to shift product formation towards ethanol and strain engineering to attain acceptable fermentation performance under challenging process conditions.

We have used directed and evolutionary approaches to further develop a strain of T. saccharolyticum previously engineered for high ethanol yield. Through the use of metabolic and transcriptional profiling, we have identified potential bottlenecks in the ethanol pathway and gained new insights into the impact of inhibitors present in the cellulose-to-ethanol process. Methods have been developed that allowed directed genetic manipulations, use of knockout and over-expression libraries, mutagenesis and growth selection to produce strains with increased performance characteristics, including ethanol production from mixed sugars at concentrations > 60 g/l.

A similar approach has been pursued for the development of C. thermocellum. Utilizing newly developed genetic tools, we have engineered the central metabolism of C. thermocellum and generated strains which do not produce lactic and acetic acid. Strain adaptation, along with genome re-sequencing, transcriptional profiling and fermentation studies have been used to develop strains demonstrating improved growth under process conditions. When combined with T. saccharolyticum, the engineered C. thermocellum enables consolidated bioprocessing of complex lignocellulosic material to ethanol.