Physiology of S. cerevisiae in continuous VHG ethanol fermentation
During CVEF, oscillatory behavior of fermentation parameters, such as biomass, cell viability, ethanol, residual glucose, and glycerol, are displayed periodically and sustained with a period of approximately 150 h at the dilution rate of 0.027 h-1 (Bai, Chen, Anderson, et al., 2004; Wang et al., 2013). In order to focus on the intracellular regulation mechanism of yeast cells during the oscillatory process, a complete representative period was shown in Figure 1, in which ethanol, glycerol, biomass, cell viability and living cells exhibited the similar changing tendencies, while residual glucose presented the opposite profile.
Considering the periodic wave characteristics, the different phases of sinusoidal function (0π, π/2, π, and 3π/2) are appropriately applied to describe the changing trends of parameters for better exploring and comprehending their correlations (Figure S1). The oscillation curve of residual glucose is configured as a 0π phase, so the profiles of ethanol and glycerol are correspondingly considered as a π phase. Because ethanol as the major product requires the consumption of glucose in proportion, it naturally follows that there is an opposite tendency between them. The parameters associated with cell growth (biomass, cell viability, and living cells) are also classified as a π phase based on the foundational knowledge that cell growth couples with the generation of primary metabolites such as ethanol. However, the changes of cell growth and viability happened prior to other parameters, reflecting a delayed response of intracellular metabolites. In addition, both high glucose and ethanol caused a synergy inhibition on viability. According to the biomass curve, five representative sampling points (P1-5 in Figure 1) were selected for subsequent metabolic profile analysis and transcriptomic analysis.