| High cost is the biggest challenge in fuel ethanol production, in which energy consumption comprises about 30%, the second largest only after feedstock consumption, and thus saving energy consumption is one of the most effective strategies to make fuel ethanol more economically competitive. Very high gravity (VHG) fermentation can significantly increase ethanol titer in the fermentation broth, which not only saves energy consumption for the downstream distillation but also for the waste distillage treatment due to its significant reduction. However, high ethanol concentration severely inhibits yeast cells, and stuck fermentation frequently occurs, with more sugars unfermented and ethanol yield compromised correspondingly. Therefore, exploration of the mechanism underlying the ethanol tolerance of yeast cells is one of the prerequisites for developing effective VHG ethanol fermentation process.In the present work, the effect of nutrients including (NH4)2SO4, K2HPO4, vitamins and bivalent metal ions from MgSO4·7H2O, CaCl2·2H2O, FeSO4·7H2O, CoCl2·6H2O, ZnSO4·7H2O and MnCl2·4H2O on the growth of yeast cells, their ethanol tolerance and production was investigated using a defined medium and the self-flocculating yeast SPSC01. The experimental results indicated that the supplementation of (NH4)2SO4, K2HPO4 and vitamins stimulated the growth of yeast cells and improve their ethanol tolerance and production, correspondingly, so did the supplementation of Mg2+, Ca2+ and Zn2+. Although the supplementation of Mn2+, Fe2+ and Co2+ had no effect on the ethanol tolerance, Co2+ improved the ethanol production, but Mn2+ had no such an effect, and Fe2+ affected it negatively. The supplementation of the seven nutrients was further optimized by the uniform design, and regression equations for ethanol tolerance and production predictions were developed. The maximum ethanol tolerance characterized by the viability of yeast cells suffered from ethanol shock treatment and ethanol production were estimated to be (91.1±1.74)% and 47.90±0.95 g l-1, respectively, which were validated by the experimental results of (90.2±0.89)% and 47.15±0.85 g l-1, correspondingly. Meanwhile, compared with the maximum ethanol tolerance and production of 83.5% and 43.70 g l-1 achieved via the single factor optimization, improvement in the two parameters were observed, indicating the synergistic role of these factors on the yeast cells.Since the supplementation of Zn2+ significantly improved the ethanol tolerance and production of the yeast cells, zinc sulfate was thus supplemented at the concentrations of 0.01, 0.05 and 0.10 g l-1 to further investigate its impact on the ethanol fermentation. It was observed that Zn2+ affected the flocculation of the yeast, and improved its ethanol tolerance, thermal tolerance and ethanol production, indicating that a process engineering strategy for improving VHG ethanol fermentation could be developed based on the Zn2+ supplementation. Moreover, intracellular accumulation of Zn2+ stimulated the accumulation of ergosterol and trehalose, two components that are beneficial for yeast cells to protect from ethanol inhibition. In comparison with the control without Zn2+ supplementation, ethanol production was improved by 8.4%, thermal and ethanol tolerance by 20.0%, while glycerol production decreased by 42% under 0.05 g l-1 zinc sulfate supplementation conditions.Furthermore, the impact of the size of the yeast flocs on the ethanol fermentation was studied by controlling their size at 100,200 and 300μm, which was characterized by their average chord monitored by the focused beam reflectance measurement. For the yeast flocs with the size of 300μm,0.05 g l-1 zinc sulfate was supplemented, with no Zn2+ supplementation as the control. The experimental results illustrated that the yeast flocs with the size of 300μm exhibited maximum ethanol production, ethanol tolerance and glucose consumption, with 110.0 g l-1 ethanol produced,58.0% viability remained for the yeast cells after 20% (v/v) ethanol shock for 5 h and glucose uptake rate of 286.69 C-mmol l-1 h-1. Intracellular ergosterol and trehalose accumulations were improved by 71.6% and 96.5%, respectively, for the yeast flocs with the size of 300μm and supplemented with 0.05 g l-1 zinc sulfate, but glycerol decreased by 37.6%. No pyruvate accumulation was observed in the Zn2+ supplementation group, and ethanol production was increased by 9.1% with 31.0% improvement of the yeast cell viability. And in the meantime, carbon fluxes at the node of glucose uptake, ergosterol, trehalose and ethanol were enhanced by 5.3%,28.6%,43.3% and 9.1%, correnpondingly.Moreover, the retention of the self-flocculating yeast was improved for the yeast flocs with relatively large size and higher biomass concentration was achieved within the bioreactor, contributing to more glucose consumed and carbon flux to ethanol production. The decrease in the fluxes to the growth of the yeast cells such as DNA, RNA, protein and lipid biosynthesis as well as the increase of ATP consumption for the maintenance indicated that improved ethanol production resulted from Zn2+ supplementation and the increased yeast floc size raised more energy burden on the yeast cells, and glucose uptake of the yeast flcos was compromised under VHG conditions, making the improvement of glucose uptake rate the key to improve VHG ethanol fermentation.
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