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The recently engineered Geobacillus thermoglucosidasius strain is an industrially potent thermophilic ethanologen. We employ a systematic approach to improve our understanding of the fermentation process using cellobiose as the substrate. Dynamic metabolic flux analysis clearly shows that the fluxes from pyruvate to lactate and formate are both strictly constrained throughout the process and that both the maximum ethanol yield (0.46C/C) and the maximum specific productivity (19.8mmolC (gDCW)h) occur at late-exponential growth phase. Accordingly, extreme pathway analysis reduces the metabolic network into a macro reaction scheme, on which a dynamic metabolic model is built. The model is validated with experimental data, parameters are identified with confidence intervals, and global sensitivity analysis (Sobol' method) is performed. Model-based optimization predicts that ethanol productivity could increase from 34.2 in a typical batch process to 55.3mmolLh in an optimum fed-batch process with higher ethanol yield. Furthermore, the optimal operating regime was identified to be continuous fermentation process with gas stripping, in which a high ethanol productivity of 113mmolLh, i.e., 26.8mmolC (gDCW)h, corresponding to 90.2% of the maximum theoretical ethanol yield could be achieved.
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