(274f) Metabolic Network Analysis of A Novel Ethanol-Tolerant Thermophilic Bacterium Geobacillus Thermoglucosidasius M10EXG for Ethanol and Lactate Fermentation

A recently discovered thermophilic bacterium, Geobacillus thermoglucosidasius M10EXG (Gth M10EXG), is a facultative anaerobe that ferments a range of C5 and C6 sugars and is tolerant to high ethanol concentrations (10%v/v). These properties make it an attractive ethanologen for bioethanol production. We have investigated the central metabolism of this bacterium using 13C-based flux analysis to provide insights into the physiological properties of this extremophile, as there is no genome information available for Gth M10EXG. Glucose metabolism in Gth M10EXG proceeds via glycolysis, the pentose phosphate pathway, the glyoxylate shunt, and the TCA cycle; no Entner-Doudoroff pathway activity was evident. Anaplerotic reactions (including pyruvate carboxylase and phosphoenolpyruvate carboxykinase) were active and together form a futile cycle. When growth conditions were switched from aerobic to micro-aerobic conditions, flux (based on a normalized glucose uptake rate of 100 units/gm biomass∙hr) through the TCA cycle and oxidative pentose phosphate pathway was reduced from 64 to 25 and from 29 to 18, respectively. This was consistent with down-regulation of anaplerotic fluxes via pyruvate carboxylase and phosphoenolpyruvate carboxykinase. The carbon flux was directed to ethanol, L-lactate (>99% optical purity), acetate and formic acid. Under fully anaerobic conditions, Gth M10EXG used a mixed acid fermentation process and exhibited a maximum ethanol yield of 0.32±0.05 mol /mol glucose. An in silico flux balance model was used to determine effect of extra-cellular and intra-cellular metabolite fluxes on ethanol production by Gth M10EXG. The results indicate that mixed acid production from Gth M10EXG significantly reduces ethanol production by this organism.