Discussion
Transcriptomic and metabolomic analysis were employed in this study to address the cellular response of yeast cells during sustained VHG oscillation. In a typical sinusoid period, P1 was consistent with P5 due to almost no difference in gene expression observed between them. Thus, the VHG oscillation is regarded as a closed cycle. The end point P5 is essentially just the start point P1 for the next period.
Among all kinds of stresses that yeast cells encounter in CVEF, dynamic ethanol inhibition was highlighted in previous studies and recognized to be responsible for oscillation(Wang et al., 2013). Expression level of most genes referring to anabolism, such as ribosome synthesis pathway, were up-regulated from phase P1 to P3, while down-regulated from phase P3 to P5, presented as an 0π phase pattern, suggesting that cellular anabolism might be regulated by the similar factors. Further, combined with the changing of ethanol, which presented as a π phase pattern, this suggests that cellular anabolism might be modulated by ethanol inhibition.
A certain critical ethanol concentration (Ecrit) was speculated at approximately 50 g/L (Wang et al., 2013). If the ethanol concentration exceeded the Ecrit, the cell proliferation of the yeast culture was significantly inhibited while at the same time the biomass level was decreasing due to continuous dilution. However, most cells still survived and produced ethanol without cell proliferation, thus the ethanol in the broth would climb gradually to the maximum concentration until yeast cells lost fermentation ability. Then ethanol in the broth would decrease due to continuous dilution, until the ethanol inhibition imposed on yeast cells was attenuated. When the ethanol concentration declined below Ecrit, the yeast cells would gradually revive the abilities of proliferation and fermentation, such as ribosome synthesis and the biomass and ethanol levels in the broth accumulated gradually. Unless the specific rate of ethanol production and biomass formation passed over the dilution rate (D=0.027 h-1), biomass and ethanol in broth would decrease for a long time. As soon as the yeast cells proliferated and the ethanol production rate surpassed the dilution rate, the ethanol concentration increased to a higher level. The ethanol inhibition imposed on yeast cells would then become stronger and cell proliferation and fermentation would subsequently be inhibited and another VHG oscillation period would be initiated. Hence, the sustained VHG oscillation is a result of the competition of cell growth, ethanol formation and ethanol inhibition.
The concentration of metabolites in the glycolytic pathway exhibited a cyclical oscillation behavior. There was a π/2 phase difference between the curve of metabolites in the energy consumption stage and the production stage. The generation of this oscillation behavior may originate from phosphofructokinase (PFK), indicating that the VHG oscillation behavior may be the result of the combined effect of intracellular regulation and extracellular ethanol stress in yeast cells.
This work has analyzed the intracellular responses of the yeast cells to the dynamic ethanol inhibition during VHG oscillation. The enhancement of ethanol tolerance of the strain could alleviate the stress of ethanol on cells, thus helping to attenuate the oscillation effect in CVEF. In addition, further transformation of related metabolic pathways by genetic engineering may weaken the oscillation behavior at the cellular molecular level, which may enable VHG continuous ethanol fermentation technology to meet industrial production requirements.