Understanding Substrate Transport and Metabolism in Clostridium thermocellum to Improve Biofuel Production

Abstract

As a cellulolytic specialist microbe, Clostridium thermocellum primarily ferments cellulose and its breakdown products. However, the full breadth of its substrate range remains poorly understood. In this work, we investigated the ability of C. thermocellum to transport and metabolize a diverse range of alternative carbon substrates. We performed tetrazolium dye assays to assess the ability of wild-type (WT) C. thermocellum (strain LL1004) to transport and metabolize 190 different substrates and identified 18 positive hits and 27 false positives. These findings guided the selection of 22 substrates for batch fermentations of WT on 10 g/L alternative substrate plus 1 g/L cellobiose. We identified eight substrates that were either fully or partially consumed (cellobiose, glucose, fructose, sorbitol, arbutin, turanose, pyruvate, glucose-6-phosphate), two that were interconverted (malate, fumarate), seven that may undergo conversion to unidentified fermentation products (xylose, sorbose, tagatose, galactose, mannose, rhamnose, fucose), and five that were not consumed (glycerol, acetate, lactate, propionate, xylitol). Sorbitol and pyruvate, which are more and less reduced than cellobiose, respectively, were selected for further analysis of their effects on redox balance and product formation. Batch fermentations on sorbitol and pyruvate demonstrated that ethanol production in C. thermocellum may be limited in part by the availability of reducing equivalents. Collectively, this work demonstrates the utility of alternative carbon substrates as a tool for perturbing metabolism, redox balance, and fermentation product distributions to further our understanding of the metabolism of anaerobic, thermophilic bacteria such as C. thermocellum for the production of biofuels and biochemicals of interest. Importance This work has advanced our understanding of the substrate range of C. thermocellum beyond its known substrates and identified several substrates including arbutin, turanose, pyruvate, and glucose-6-phosphate that C. thermocellum can metabolize and convert to fermentation products. This work has also demonstrated the use of alternative carbon substrates as a tool for perturbing metabolism, altering redox balance, and changing fermentation product profiles to advance our understanding of a metabolic pathway of interest, such as the cellobiose to ethanol production pathway in C. thermocellum. These insights can be used to inform future research aimed at engineering a strain of C. thermocellum for the production of cellulosic ethanol at high yield, titer, and rate. This experimental approach can be broadly applied to advance our understanding of the physiology and metabolism of other microorganisms and to engineer promising microbial candidates for the biomanufacturing of biofuels, biochemicals, or pharmaceuticals of interest.