(440g) Improving Ethanol Tolerance of Pichia Stipitis Via Continuous Fermentation with Cell Retention

In view of rising concerns over energy sustainability and global warming, the need for biofuels is expected to increase sharply in the coming years [1]. At present, the U.S. biofuels market is dominated by corn-derived ethanol. However, substantial future growth of fuel ethanol will depend upon developments of cellulosic ethanol processes, because cellulosic biomass is the most abundant and inexpensive renewable feedstock for ethanol production. Moreover, it is estimated that cellulosic ethanol reduces both energy input and greenhouse gas emission by over 85% compared to corn ethanol [2].

Due to its high abundance in cellulosic biomass, xylose utilization is critical for commercial cellulosic ethanol processes. To make cellulosic ethanol the long-term renewable energy source, microbes that can ferment xylose into ethanol with a high yield and productivity are needed. Among all the native xylose-fermenting yeast strains, Pichia stipitis has been shown to be the most promising one for direct high-yield fermentation of xylose without byproduct formation [3]. However, similar to other xylose-fermenting strains, P. Stipitis has limited ethanol tolerance, which is one of the reasons that hinder its industrial application for xylose fermentation.

The focus of this work is to improve P. Stipitis's ethanol tolerance. Many studies have indicated that ethanol tolerance of yeast strains can be enhanced by cell immobilization [4-7]. It has been suggested that the improvement caused by immobilization is due to the enhanced hydration layer stability resulted from attaching cells to the carrier. In this research, we propose a different explanation. We hypothesize that the ethanol tolerance enhancement associated with cell immobilization is due to adaptation. For immobilized cells, the ethanol concentration in the small vicinity of the cells is higher than the bulk concentration because of the mass transfer resistance associated with the carrier. Therefore, immobilized cells are exposed to higher ethanol concentration compared to free floating cells, and become adapted to higher ethanol concentration as the ethanol concentration gradually increases during the fermentation process. In addition, our hypothesis is supported by the following observations: 1) it is reported that after cells are removed from the carrier, they still maintain the improved ethanol tolerance [8]; 2) it is found that cell immobilization improves ethanol tolerance in both yeast and bacteria [7,8], even though yeasts and bacteria have completely different membrane structures.

In this work, we test our hypothesis using continuous fermentation with cell retention. Such pseudo-continuous fermentation provides an ideal environment for cell adaptation, as it enables us to increase the ethanol concentration in the bulk solution gradually which allow cells to adapt to the harsh environment. The effect of cell retention, ethanol concentration and adaptation time, etc., on ethanol tolerance improvement are studied and compared with the effect of cell immobilization in batch fermentation.

Key words:

Cellulosic ethanol, xylose fermentation, ethanol tolerance, Pichia stipitis, adaptation

Reference

1. U.S. Congress (2007), Energy independence and security act of 2007.

2. Farrell AE, et al. (2006), Ethanol Can Contribute to Energy and Environmental Goals, Science, 311 (5760), 506-508

3. McMillan JD (1993), Xylose fermentation to ethanol: A review, Published by National Renewable Energy Laboratory, ISBN: B0006QLMFU

4. Ciesarova Z, Domeny Z, Smogrovicova D, Patkova J, Sturdik E. (1998), Comparison of ethanol tolerance of free and immobilized Saccharomyces uvarum yeasts. Folia Microbiol 43, 55-58

5. Desimone MF, Degrossi J, D'Aquino M and Diaz LE (2002) Ethanol tolerance in free and sol-gel immobilised Saccharomyces cerevisiae. Biotechnol. Lett., 24, 1557?1559

6. Jirku V (1999), Whole cell immobilization as a means of enhancing ethanol tolerance. J. Indust. Microbiol. Biotechnol., 22, 147-151

7. KRISCH J, SZAJANI B (1997), Ethanol and acetic acid tolerance in free and immobilized cells of Saccharomyces cerevisiae and Acetobacter aceti, Biotechnology letters, 19(6), 525-528

8. Zhou B, Martin GJO and Pamment NB (2008), Increased phenotypic stability and ethanol tolerance of recombinant Escherichia coli KO11 when immobilized in continuous fluidized bed culture, Biotechnol. Bioeng., 100, 627?633