Pseudo-Continuous Fermentation Using a Novel Bioreactor to Facilitate the Study of a Co-Culture System for Ethanol Production

Keywords: bioreactor, co-culture, fermentation, ethanol production, co-fermentation, bioethanol, lignocellulose, mixed substrate, P. stipitis, S. cerevisiae

The bioethanol industry has developed rapidly in recent years to cope with the rising cost of fossil fuel. The conversion of lignocellulosic materials into ethanol has been regarded as one of the most promising renewable energy sources. This is not only because of the moderate cost and high availability of lignocellulosic biomass, but also because lignocellulosic ethanol process can greatly reduce CO₂ emission [1]. Glucose and xylose are two major components in the lignocellulosic hydrolysates. While glucose fermentation is well established, xylose fermentation remains an unsolved problem in the industrialized lignocellulosic ethanol process [2]. In existing studies of ethanolic fermentation, besides recombinant strategy, co-culture strategy has drawn much research interest in the past few decades [3-7]. However, currently there is a lack of systematic study on the properties of the co-culture system.

In this work, we focus on the co-culture system consisting of Saccharomyces cerevisiae and Pichia stipitis. One of the major difficulties of the co-culture strategy for simultaneous glucose/xylose fermentation is the inability to provide the optimal fermentation conditions for two different strains [8-10]. Most of studies on co-culture process reported that while the fermentation of glucose in the mixed substrate proceeded efficiently with a glucose-fermenting yeast strain, the fermentation of xylose with a xylose-fermenting strain often exhibited low ethanol productivities due to the differences in the fermentative regulation for ethanol production [11-15]. Another difficulty of co-culture strategy is the diauxic growth of P. stipitis due to its preferential utilization of glucose over xylose when cultured on mixed substrates [7, 11,16].

In order to address these difficulties and to improve the co-culture process efficiency, we have developed a novel bioreactor which separates the two different yeast strains, and enables different fermentative conditions for different strains. In addition, we have built a cell retention module which enables the pseudo-continuous fermentation mode, i.e. continuous fermentation with cell retention. With pseudo-continuous operation, we can completely eliminate the possible wash-out associated with continuous fermentation and can easily test different operation conditions. In this study, we show that pseudo-continuous operation using the developed bioreactor provides an effective tool to study co-culture systems, which allow us to investigate the dynamic of co-culture system for lignocellulosic ethanol production systemically.

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