Thesis Committee

Lee R. Lynd, DE. (Chair)

Margaret E. Ackerman, Ph.D.

Karl E. Griswold, Ph.D.

Philippe Soucaille, Ph.D.

Abstract

Cellulosic ethanol is a promising renewable fuel for reducing our dependence on fossil fuels.  However, current methods for producing cellulosic ethanol are energy intensive and thus come with high processing cost, in part due to cellulosic biomass’ recalcitrance to being degraded into fermentable sugars.

One low-cost strategy for producing cellulosic ethanol is consolidated bioprocessing (CBP), which involves the simultaneous degrading of cellulosic biomass and fermenting of the released sugars into ethanol by a cellulolytic, ethanol-producing microorganism, without the need for additional cellulolytic enzymes.

Clostridium thermocellum is a promising candidate organism for consolidated bioprocessing (CBP) of lignocellulosic biomass into ethanol; this thermophilic anaerobic bacterium is naturally capable of solubilizing cellulosic biomass at high rates, and can ferment the released sugars into several fermentation products including ethanol.  In recent years, there have been multiple attempts at engineering C. thermocellum to produce ethanol at high yield and titer, with varying degrees of success.

Thermoanaerobacterium saccharolyticum is a non-cellulolytic, thermophilic, anaerobic bacterium that has been successfully engineered to produce ethanol at high yield and titer.  Recent progress in understanding this bacterium’s metabolism has led to the identifying of the key enzymes that enable it to produce ethanol at high yield.  However, introducing these enzymes into C. thermocellum will first require that we have a robust and reliable genetic toolkit to engineer C. thermocellum.

This thesis seeks to re-create the ethanol producing pathway of T. saccharolyticum in C. thermocellum, first by further developing the genetic tools for C. thermocellum to enable higher throughput genetic engineering efforts, and then using them to introduce the key genes of the T. saccharolyticum ethanol producing pathway into C. thermocellum, with the aim of creating a better ethanologen strain of C. thermocellum than already exists.  This thesis will also explore the impact of replacing C. thermocellum genes that play a role in ethanol yield and titer with their T. saccharolyticum homologs in addition to the novel genes that have already been introduced.  As part of this thesis, we also hope to also gain a better understanding of the differences between T. saccharolyticum and C. thermocellum metabolism, and how these differences allow the former to produce ethanol at high yield and titer.