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.