Compared with steady state, oscillation in continuous very-high-gravity ethanol fermentation with Saccharomyces cerevisiae improved process productivity, which was thus introduced for the fermentation system composed of a tank fermentor followed by four-stage packed tubular bioreactors. When the very-high-gravity medium containing 280g l?1 glucose was fed at the dilution rate of 0.04 h?1, the average ethanol of 15.8% (v/v) and residual glucose of 1.5g l?1 were achieved under the oscillatory state, with an average ethanol productivity of 2.14g h?1l?1. By contrast, only 14.8% (v/v) ethanol was achieved under the steady state at the same dilution rate, and the residual glucose was as high as 17.1 g l?1, with an ethanol productivity of 2.00g h?1l?1, indicating a 7% improvement under the oscillatory state. When the fermentation system was operated under the steady state at the dilution rate of 0.027 h?1 to extend the average fermentation time to 88 h from 59 h, the ethanol concentration increased slightly to 15.4% (v/v) and residual glucose decreased to 7.3 g l?1, correspondingly, but the ethanol productivity was decreased drastically to 1.43 g h?1l?1, indicating a 48% improvement under the oscillatory state at the dilution rate of 0.04 h?1.

Taking continuous ethanol fermentation with the self-flocculating yeast SPSC01 under very high concentration conditions as an example, the fermentation performance of the yeast flocs and their metabolic flux distribution were investigated by controlling their average sizes at 100, 200, and 300mm using the focused beam reflectance online measurement system. In addition, the impact of zinc supplementation was evaluated for the yeast flocs at the size of 300mm grown in presence or absence of 0.05 g L 1 zinc sulfate. Among the yeast flocs with different sizes, the group with the average size of 300 mm exhibited highest ethanol production (110.0 g L 1) and glucose uptake rate (286.69 C mmol L 1 h 1), which are in accordance with the increased flux from pyruvate to ethanol and decreased flux to glycerol. And in the meantime, zinc supplementation further increased ethanol production and cell viability comparing with the control. Zinc addition enhanced the carbon fluxes to the biosynthesis of ergosterol (28.6%) and trehalose (43.3%), whereas the fluxes towards glycerol, protein biosynthesis, and tricarboxylic acid cycle significantly decreased by 37.7%, 19.5%, and 27.8%, respectively. This work presents the first report on the regulation of metabolic flux by the size of yeast flocs and zinc supplementation, which provides the potential for developing engineering strategy to optimize the fermentation system.

Yeast strains of Saccharomyces cerevisiae have been extensively studied in recent years for fuel ethanol production, in which yeast cells are exposed to various stresses such as high temperature, ethanol inhibition, and osmotic pressure fromproduct and substrate sugars aswell as the inhibitory substances released from the pretreatment of lignocellulosic biomass. An in-depth understanding of the mechanism of yeast stress tolerance contributes to breeding more robust strains for ethanol production, especially under very high gravity conditions. Taking advantage of the "omics" technology, the stress response and defense mechanism of yeast cells during ethanol fermentationwere further explored, and the newly emerged tools such as genome shuffling and global transcription machinery engineering have been applied to breed stress resistant yeast strains for ethanol production. In this review, the latest development of stress tolerance mechanisms was focused, and improvement of yeast stress tolerance by both random and rational tools was presented.

Process oscillation characterized by long oscillation period and large oscillation amplitude was observed in continuous ethanol fermentation with Saccharomyces cerevisiae under very high gravity conditions. Metabolic flux analysis was applied to the fermentation system, and the results indicated that carbon flux distributions at the metabolic notes oscillated, correspondingly, and the root reason for the process oscillation was the intracellular metabolism of yeast cells. Cell cycle analysis with the flow cytometry showed that no cell-cycle-dependent synchronization of the daughter and mother cells occurred within the duration of the oscillation, and thus different mechanism existed compared with the oscillation observed in the continuous culture of Saccharomyces cerevisiae and triggered by the synchronization of the daughter and mother cells under specific conditions. Furthermore, the overall metabolic activity of the yeast cells was examined, which was found not exactly out of phase but lag behind ethanol concentration that accumulated within the fermentation system and its inhibition on the yeast cells as well, which supported the mechanistic speculation for the process oscillation: the lag response of yeast cells to ethanol inhibition.

Yeast flocculation has been used in the brewing industry to facilitate biomass recovery for a long time, and thus its mechanism of yeast flocculation has been intensively studied. However, the application of flocculating yeast in ethanol production garnered attention mainly in the 1980s and 1990s. In this article, updated research progress in the molecular mechanism of yeast flocculation and the impact of environmental conditions on yeast flocculation are reviewed. Construction of flocculating yeast strains by genetic approach and utilization of yeast flocculation for ethanol production from various feedstocks were presented. The concept of self-immobilized yeast cells through their flocculation is revisited through a case study of continuous ethanol fermentation with the flocculating yeast SPSC01, and their technical and economic advantages are highlighted by comparing with yeast cells immobilized with supporting materials and regular free yeast cells as well. Taking the flocculating yeast SPSC01 as an example, the ethanol tolerance of the flocculating yeast was also discussed.