(279f) High-Efficient Butanol Production from Non-Detoxified Corn Stover Hydrolysate By Strain Development and Membrane Separation Using Clostridium Acetobutylicum

You-Duo Wu¹*and Chuang Xue¹

¹School of Bioengineering, Dalian University of Technology, Dalian, China

Corn stover (CS) is evaluated as the most favorable candidate feedstock for butanol production via acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum. By independent acid pretreatment and enzymatic hydrolysis, fermentable sugars were released, of which glucose was naturally utilized as the most preferred carbon source by C. acetobutylicum. However, the ABE fermentation using corn stover hydrolysate (CSH) without detoxification is typically limited to poor sugars utilization, butanol production and productivity. In the presence of pretreatment-derived inhibitors, the intracellular ATP and NADH involved in cell growth, solventogenesis initiation and stress response, are exceedingly challenged owing to disrupted glucose phosphotransferase system (PTS). Therefore, there is a necessity to develop effective engineering approaches to overcome these limitations for high-efficient butanol production from CSH without detoxification.

PTS-engineered C. acetobutylicum strains were constructed via overexpression and knockout of gene glcG encoding glucose-specific PTS IICBA, which pleiotropically regulated glucose utilization, cell growth, solventogenesis and inhibitors tolerance. The PTS^GlcG-overexpressing strain utilized ~60 g/L glucose within 28 h, reaching a butanol productivity of 0.46 g/L/h, compared to that of 0.16 g/L/h with the PTS^GlcG-deficient strain. During CSH culture without detoxification or nutrients addition, the PTS^GlcG-overexpressing strain exhibited desirable inhibitors tolerance and solventogenesis compared to the control and PTS^GlcG-deficient strains, respectively. As a result of glucose and 10 g/L CaCO₃ addition into CSH, butanol production and productivity were maximized to 12.5 g/L and 0.39 g/L/h. These validated improvements on the PTS^GlcG-overexpressing strain were attributed to not only amplified carbon flux but boosted supplies of intracellular ATP and NADH at exponential growth phase compared to those of the control strain during glucose culture. Furthermore, the fermentation integrated with membrane separation was carried out for butanol removal to eliminate its toxicity to cells. This in situ process could enhance butanol productivity and yield due to the enhanced activity of cells during butanol fermentation.

In a word, combined with the application of membrane separation, the PTS^GluG regulation could be an effective engineering approach for high-efficient ABE fermentation from lignocellulosic hydrolysates without detoxification or wastewater generation, providing fundamental requirements for economically sustainable butanol production with high productivity.

* Corresponding author, No 2 Linggong Road, School of Bioengineering, Dalian University of Technology, Dalian 116024, China, Tel: +86-411-84706625, Fax: +86-411-84706625, E-mail: wuyouduo@dlut.edu.cn.