Metabolic inhibition of Clostridium thermocellum by pentose sugars

The native hydrolytic machinery of the thermophilic anaerobe Clostridium thermocellum has marked this bacterium as a leading candidate for the production of lignocellulosic biofuels through consolidated bioprocessing. Despite its unique plant cell-wall deconstructing capabilities, the limited substrate utilization potential of the strain, particularly in regards to its inability to ferment pentose sugars, represents a major obstacle to overcome before its industrial deployment. In this study, we not only confirm the inability of C. thermocellum to ferment pentose sugars, but also provide novel data illustrating that increasing concentrations of C5-sugars lead to corresponding levels of inhibited growth with an IC₅₀ of ~15 g/L. Further, through the use of ¹³C-xylose based metabolomics analyses, we offer evidence that xylose can act as a redox-reactive compound susceptible to transformation by C. thermocellum to form xylitol and/or xylulose. Expansion of our study beyond the use of the model substrate xylose to include xylo-oligomers of varying chain lengths further confirms the potential for xylan hydrolysis products to metabolically inhibit C. thermocellum. While the level of inhibition was influenced by xylo-oligomer chain length, the enantiomeric configuration of pentose sugars proved to be even more influential suggesting an enzymatic basis for the observed inhibition. Guided by RNA-seq analyses, attempts to identify and understand the specific biological basis for inhibition have been explored and detailed discussions regarding these mechanisms will be undertaken. Further, evidence supporting the existence of a potential quorum-sensing system in C. thermocellum will also be presented.