(608c) A Novel Co-Culture System for Pseudo-Continuous Ethanolic Fermentation From Lignocellulosic Sugars

Lignocellulosic biomass is an attractive sustainable carbon source for ethanolic fermentation process. However, the lack of microorganism which can efficiently ferment all sugars derived from lignocellulosic biomass is one of the key challenges in its commercial utilization for biofuel. To achieve efficient utilization of glucose/xylose mixtures for ethanol production, many genetic and metabolic engineering of wild-type strains have been studied to develop a single recombinant strain which can simultaneously ferment both glucose and xylose to produce ethanol. As an alternative, co-culture strategy has been proposed to simultaneously consume glucose and xylose.

However, efficient ethanol production by conventional co-culture systems faces several difficulties and the major ones are the competition for dissolved oxygen between the microbes, the diauxic behavior of xylose-fermenting strain (i.e., they cannot use xylose in the presence of glucose) and their low ethanol tolerance. In addition, very limited research has been done to investigate the dynamic properties of co-culture systems. Since the interactions between two microorganisms (glucose-fermenting and xylose-fermenting strains) are complex due to different assimilation pathways and regulatory mechanisms, it is important to develop new approaches to help understand and explore the dynamic behaviors of the co-culture system.

In this work, we have developed a novel co-culture system for the efficient and simultaneous conversion of mixed glucose and xylose to ethanol by Saccharomyces cerevisiae (S. cerevisiae) and Scheffersomyces stipitis (S. stipitis). The two-chambered bioreactor was designed and constructed in-house by placing a microporous filter membrane between the two halves of the bioreactor. This configured co-culture system not only enables the confinement of each strain to allow the separate control and monitor of the cell growth, but also allows the independent control of optimal oxygen conditions of both strains. At the same time, the system allows the exchange of the culture medium and extracellular metabolites between the two chambers. For example, it is possible to maintain the oxygen supply conditions suitable for each strain with accurately controlled oxygen transfer rate (OTR) by adjusting the air and nitrogen gas flow rates into each chamber using an in-house developed gas-mixing apparatus. Furthermore, an innovative fermentation scheme of pseudo-continuous fermentation (i.e., continuous fermentation with cell retention) in the novel co-culture system allows us to use a wide range of dilution rates without worrying about the cell washout and to prevent the diauxic kinetics caused by the xylose catabolite repression associated with S. stipitis. It is shown that the novel co-culture system is an effective way to study the dynamic properties of co-culture system and the metabolic interactions between two co-culture strains. With this novel system, the effects of various fermentative conditions such as dilution rate, OTR, temperature and pH on co-culture dynamics have been systematically investigated and will be discussed in detail.