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.