Characterization and Amelioration of Filtration Difficulties Encountered in Metabolomic Studies of Clostridium thermocellum at Elevated Sugar Concentrations

Clostridium thermocellum, a promising candidate for consolidated bioprocessing, has been subjected to numerous engineering strategies for enhanced bioethanol production. Measurements of intracellular metabolites at substrate concentrations high enough (>50 g/L) to allow the production of industrially relevant titers of ethanol would inform efforts toward this end but have been difficult due to the production of a viscous substance that interferes with the filtration and quenching steps during metabolite extraction. To determine whether this problem is unique to C. thermocellum, we performed filtration experiments with other organisms that have been engineered for high-titer ethanol production, including Escherichia coli and Thermoanaerobacterium saccharolyticum. We addressed the problem through a series of improvements, including active pH control (to reduce problems with viscosity), investigation of different filter materials and pore sizes (to increase the filtration capacity), and correction for extracellular metabolite concentrations, and we developed a technique for more accurate intracellular metabolite measurements at elevated substrate concentrations.

Automated summary

In order to address the difficulties in measuring intracellular metabolites in C. thermocellum for bioethanol production, improvements were made including active pH control, investigation of different filter materials and pore sizes, and correction for extracellular metabolite concentrations. This resulted in the development of a technique for more accurate intracellular metabolite measurements at high substrate concentrations.