2-05: Insights into the physiology of thermophilic microbial cellulose utilization

Commercially viable cellulosic ethanol is likely to be produced by large-scale microbial solid substrate fermentations, with Consolidated Bioprocessing (CBP) as the most cost-effective approach. However, the physiology of microbial cellulose utilization on solid substrates as applied in CBP is poorly understood. The main obstacle is the lack of methods that can discriminate between substrate and microbes. Clostridium thermocellum is a model CBP organism that can convert crystalline cellulose to ethanol, whereby the cells utilize cellulose by adhering and colonizing the substrate particles, and therefore cannot be distinguished from the substrate by conventional methods. This phenomenon makes accurate evaluations of solid substrate cultures difficult.

We discuss the development and application of methods based on elemental analysis and real-time fermentation data for C.thermocellum that enable us to accurately follow the growth of biomass and utilization of substrate.  Results include a) cultures grown on solid substrate do not exhibit a constant specific growth rate, and b) the rate of cellulose utilization is a second order process dependent of both the substrate and biomass concentration. By using parameters determined by this methodology we can now predict the amount of cellular biomass from the residual substrate concentration.

Furthermore we show results of C.thermocellum growing on high solids concentrations and ‘real-world’ substrate. When cultured at 80-g/L Avicel (~20% solids loading equivalent) C.thermocellum was observed to stop growing during fermentation and switch to uncoupled metabolism to utilize the remainder of the substrate. Our studies provide valuable insights for strain-development and opportunities of C.thermocellum as thermophilic CBP platform.