METABOLITE ANALYSIS OF CLOSTRIDIUM THERMOCELLUM USING CAPILLARY ELECTROPHORESIS BASED TECHNIQUES

Clostridium thermocellum is a thermophilic bacterium that converts biomass to ethanol directly; however, high sensitivity of this bacterium toward ethanol limits its commercial utility. To elucidate the effect of ethanol on the growth of this bacterium a metabolite analysis of C. thermocellum was performed. The hypothesis of the project was that exogenous ethanol alters the metabolite profile of C. thermocellum. For metabolite analysis, capillary electrophoresis-electrospray ionization-mass spectrometry method (CE-ESI-MS) was developed due to highly polar and charged nature of metabolites. To increase the sensitivity of CE-ESI-MS, several parameters at the ESI interface were optimized. The application of 50% isopropanol as a sheath liquid increased sensitivity for metabolite analysis dramatically. Trimethylamine acetate (pH 10) was used as background electrolyte (BGE) due to its ability to separate the structural isomers of glucose phosphate. For metabolite sample preparation, novel methods for quenching and CE compatible metabolite extraction protocols were developed. Newly developed protocols were applied to metabolite analysis of wild type (WT) and ethanol adapted (EA) strains of C. thermocellum grown in batch cultures. Significant differences were found in key intracellular metabolites such as NAD+ and pyruvic acid. Intracellular concentrations of NAD+ were low in EA cells compared to WT cells and pyruvic acid was only detected in EA cells. To further understand the effect of ethanol on metabolite fluxes, WT and EA cells were grown in increasing concentrations of ethanol and the metabolite profile for each ethanol treatment was obtained. Significant changes were found in intracellular metabolite concentrations. Metabolic data showed that the glycolysis process in WT cells was obstructed due to exogenous ethanol which was evident from accumulation of G6P. On the other hand, no such accumulation of G6P was observed in the EA strain; however pyruvate began to accumulate in EA strain. These changes in intracellular metabolite concentrations due to perturbation of exogenous ethanol supported the hypothesis. Also, this investigation revealed a correlation between ethanol and metabolite profile changes and was able to explain a possible mechanism of growth inhibition of C. thermocellum which will certainly help genetic engineers to develop superior strains of C. thermocellum for commercial cellulosic ethanol production