Introduction
As the demand for oil further increases, the diversification of fuel
supply through alternative energy sources such as fuel ethanol has
become part of the national energy policy in many countries (Kircher,
2015). Although cellulosic ethanol is widely regarded as a promising
liquid fuel (Liu et al., 2019), a lack of substantive breakthroughs in
core technologies and low economic feasibility is still a major
limitation. This is a result of complex pretreatment requirements,
inhibitory compounds in the fermentation broth, the cost of cellulases,
and inefficient pentose metabolization by the fermentation strain (Han
et al., 2020). In addition, achievable cellulosic ethanol titers
(3%-6% v/v) are lower than starch-based ethanol titers (10%-12% v/v)
which significantly increases energy cost for distillation and stillage
treatment (Bai, Anderson, & Moo-Young, 2008).
Very high gravity (VHG) fermentation with ≥25% w/v substrate improves
ethanol titer (≥15% v/v) with concomitant savings in energy consumption
(Puligundla, Smogrovicova, Obulam, & Ko, 2011). VHG ethanol
fermentation is usually practiced via labor-intensive and
time-consuming batch operation, which is not suitable for production at
large scale . On the other hand, continuous VHG ethanol fermentation
(CVEF) could be applied in industry, but often trigger a sustained
oscillation of residual glucose, ethanol, and biomass, leading to an
undesirable unstable process (Bai, Ge, Anderson, & Moo-Young, 2009).
The toxicity of ethanol to the yeast S. cerevisiae is a primary
factor limiting titer and productivity. Ethanol tolerance is related to
the fluidity, structure, and composition of the plasma membrane, and the
levels of unsaturated fatty acids, ergosterol, amino acids, inositol,
heat shock proteins (HSPs), ATPase and the storage carbohydrates (Lam,
Ghaderi, Fink, & Stephanopoulos, 2014).
Attenuating oscillation has been attempted by engineering strategies.
Employment of three tubular bioreactors in series following a stirred
tank bioreactor packed with intalox ceramic saddle packing enhanced the
yeast tolerance to ethanol through step-by-step adaption and cell
immobilization (Bai, Chen, Anderson, & Moo-Young, 2004). Gas stripping
with nitrogen or fermentation exhaust gas directly decreased the ethanol
in the broth that help yeast cell to relieve ethanol toxicity (Wang,
Zhao, Xue, & Bai, 2013). These approaches effectively attenuated
oscillations and achieved quasi-steady-states in ethanol fermentation.
Periodic behavior has been investigated in eukaryotic and prokaryotic
organisms (Panda, 2016). S. cerevisiae exhibits various modes of
oscillatory behavior both in cell-free extract and during continuous
culture (Bai et al., 2009; Chin, Marcus, Klevecz, & Li, 2012) in the
form of glycolytic oscillation with a period of about 1 min, respiratory
oscillation with a period of 40-60 min, and cell cycle oscillation with
a period of 2-45 h (Richard, 2003). The glycolytic oscillation is
mediated by glycolytic intermediates, and the allosteric properties of
phosphofructokinase (PFK) and the positive feedback exerted on it were
assumed to be responsible for periodic operation (Gustavsson et al.,
2014; Olsen, Stock, & Bagatolli, 2020). The respiratory oscillation,
monitored by dissolved oxygen, oxygen uptake rate (OUR), and carbon
dioxide evolution rate (CER), was modulated by sulfate assimilation,
ethanol degradation and respiration (Patnaik, 2003; Tu et al., 2007).
The cell cycle oscillations, synchronized with the cell division cycle,
were represented by extracellular variables (OUR, CER, glucose, ethanol,
and biomass), intracellular variables (storage carbohydrates), and cell
cycle related variables (budding index and cell size distribution)
(Ewald, Kuehne, Zamboni, & Skotheim, 2016).
Although dynamic ethanol inhibition has been proved to be a key factor
in the induction of oscillatory behavior (Wang et al., 2013), the detail
of yeast cells response remains a fundamental and unanswered question.
In this study, a systematic approach via transcriptomic and metabolomic
analysis was carried out, with particularly focus on the cellular events
of S. cerevisiae under CVEF and cellular metabolism affected by
the fermentation parameters. A way to comprehend an unsteady state of
fermentation in such a complex metabolic oscillation is demonstrated.