Thesis Committee

Lee R. Lynd, Ph.D. (Chair)

Margaret E. Ackerman, Ph.D.

Xiongjun Shao, Ph.D.

Abstract

Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are bacteria under investigation for production of biofuels from plant biomass.  T. saccharolyticum has been engineered to produce ethanol at high yield (>90% of theoretical) and titer (>70 g/l).  Efforts to engineer C. thermocellum have not, to date, been as successful, and efforts are underway to transfer the ethanol production pathway from T. saccharolyticum to C. thermocellum.  One potential challenge in transferring metabolic pathways is the possibility of incompatible levels of nicotinamide cofactors.  These cofactors (NAD⁺, NADH, NADP⁺ and NADPH) and their oxidation state are important in the context of microbial redox metabolism.  We have directly measured the concentrations and reduced: oxidized ratios of these cofactors in a number of strains of C. thermocellum and T. saccharolyticum by using acid/base extraction and enzymatic assays.  We found that cofactor ratios are maintained in a fairly narrow range, regardless of the metabolic network modifications considered.  Also, these ratios are similar in both organisms, which is a relevant observation in the context of transferring the T. saccharolyticum ethanol production pathway to C. thermocellum.