Relating fermentation pathways to energy conservation in lignocellulolytic clostridia and related organisms

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A substitute for petroleum-based liquid transportation fuel is plant-based ethanol. An abundant, albeit recalcitrant, source of fermentable sugar for ethanol production is lignocellulose derived from plant biomass (straw, wood, etc.). Present methods involve complex pre-treatments to produce yeast-fermentable sugar. An alternate consolidated bioprocess can use cellulose-fermenting Clostridium thermocellum to produce ethanol directly from lignocellulose. Metabolic modelling has been an issue because of its “atypical” glycolytic pathway, which uses GTP and pyrophosphate along with ATP, and lacks pyruvate kinase. It is also a strict anaerobe that can hydrolyse but not ferment hemi-cellulose. These issues may be resolved if partnering with the xylan-fermenting C. stercorarium and the air-tolerant cellulose degrading C. straminisolvens. Biochemically, however, these latter organisms are poorly characterized. The goal of this program is to understand the energy conservation pathways in these 3 related organisms. In achieving its goal, this program will train 2 Ph.D., 2 M.Sc. and 5 undergraduate HQP. The objectives are: A) To define the roles of the enzymes of pathways of central metabolism, especially with respect to the energy cofactors used (GTP, ATP, pyrophosphate) in order to better understand their distinctive metabolic roles. B) To understand interactions of these organisms when grown in co-cultures on lignocellulose as the carbon source. We propose to complete the characterization of energy conservation associated enzymes of glycolysis in C. thermocellum started in the previous Discovey grant. Growth-condition-specific expression-differences of key gene-products associated with "atypic" glycolysis will be determined by RTqPCR methods, as well as interplay of the different energy molecules. Such data will lead to robust metabolic models for cellulose fermentation. A similar approach will the used for C. stercorarium and C. straminivolvens to confirm similarities and highlight differences. Expression pattern changes when these organisms interact as mixed biofilms on solid cellulosic substrates will be studied using proteomic approaches. Their spatial distribution will be observed using fluorescent-in situ-hybridization methods. One hypothesis is that C. stercorarium may be pelagic because of the different architecture of their surface cellulases, while C. thermocellum and C. straminisolvens, which both carry cellulosomes may be attached. These experiments will also be performed using Methanothermobacter thermoautotrophicus as a terminal hydrogen acceptor associated with cellulose degrading environments, as well as Caldibacillus debilis, a facultative aerobe also associated with C. thermocellum in the environmental. The metabolic understanding generated through this work will lead to developing organisms and fermentation strategies that are expected to improve the efficiency of ethanol production from cellulosic biomass for the Green Economy.