Oscillatory Behavior In Very-high-gravity Continuous Ethanol Fermentation By Saccharomyces Cerevisiae

Very-high-gravity (VHG) fermentation can significantly improve ethanol titer to save energy consumption for ethanol distillation, and in the meantime reduce stillage discharge to save energy consumption for stillage treatment, which has been practiced in batch operation that is labor-intensive and time-consuming, and not suitable for fuel ethanol production at large scale, making it urgent for developing continuous VHG ethanol fermentation systems. However, the sustained oscillation of process parameters including residual glucose, ethanol and biomass concentrations was observed during continuous VHG ethanol fermentation by Saccharomyces cerevisiae. which not only compromised ethanol production, but also affected the distillation process. In this study, process engineering strategies, metabolic profiling and transcriptomic analysis were applied to explore mechanisms underlying this phenomenon.During continuous VHG ethanol fermentation, yeast cells are suffering from ethanol inhibition and/or osmotic stress, which are associated in nature. The impact of ethanol inhibition on the process oscillation was studied by supplementing exogenous ethanol into the low-gravity (LG) medium containing-100g/L glucose, and experimental results indicated that steady state observed with continuous ethanol fermentation under the LG medium condition was disrupted, and process oscillation was triggered when30g/L ethanol was supplemented, which was augmented as ethanol supplementation was increased to50g/L. The steady state was restored when ethanol supplementations were stopped. In addition, when N2gas stripping was incorporated into the continuous VHG ethanol fermentation system to strip off ethanol produced during the fermentation, process oscillation was attenuated. As ethanol supplementation was increased to70g/L, no process oscillation was observed due to severe inhibition of ethanol in S. cerevisiae. On the other hand, non-fermentable xylose for S. cerevisiae was supplemented into the LG medium to simulate the osmotic stress under the VHG fermentation condition, no process oscillation was observed, although the osmotic stress inhibited yeast growth.The impact of ethanol tolerance with yeast strains and nutritional component of medium on the process oscillation was also investigated. Sustained oscillation was observed in continuous VHG ethanol fermentation with ethanol-tolerant strains S. cerevisiae BHL01and S. cerevisiae4126, but quasi-steady state with more residual glucose and less ethanol produced was developed with less ethanol-tolerant strain S. cerevisiae288c. During continuous VHG ethanol fermentation with S. cerevisiae4126. sustained oscillation was observed with the YPD medium supplemented with yeast extract and peptone, while damped oscillation and consequently quasi-steady state with more residual glucose and less ethanol were developed with the synthetic medium or under anaerobic conditions. These experimental results further validated that ethanol inhibition in yeast cells was the main reason for the process oscillation with continuous VHG ethanol fermentation, which was nutrition-and energy-dependent, and associated with ethanol tolerance of yeast strains as well. Furthermore, continuous VHG ethanol fermentation with the ethanol-tolerant strain S. cerevisiae BHL01was coupled with the off-gas stripping to alleviate ethanol inhibition, residual glucose less than0.10g/L and total ethanol of110.87g/L were achieved for the fermentation system, making its ethanol productivity improved to2.99g/L/h, providing a strategy for process development.In order to explore molecular mechanism underlying the process oscillation, five samples were collected within an intact oscillation period, and27intracellular metabolites and transcriptome of yeast cells were analyzed. The treatment with cold methanol (CM) and cold chloroform-methanol-buffer (CMB) was applied to metabolism quenching and extraction of intracellular metabolites for LC-MS/MS analysis with the select reaction monitor (SRM) mode and acetonitrile and20mM ammonium acetate at pH6.60as eluent.The concentrations of GLC, G6P and F6P associated with energy-consuming in the glycolytic pathway and the expression levels of HXT1, HXT3, HXK2and GLK1, encoding glucose transporter and hexokinase, respectively, were characterzed by the V profile, and their minimal levels were observed at maximal ethanol gradient, suggesting that glucose utilization in yeast cells was regulated by ethanol gradient in the continuous VHG fermentation system. However, the concentrations of GAP, G3P and PYR with energy-producing and FBP produced by the phosphorylation of fructose catalyzed by phosphofructokinase (PFK) showed the N profile, with1/4-period difference to the profile of metabolites with energy-consuming, indicating that metabolic oscillation existed in yeast cells and regulated by PFK. The expression levels of PDC1and ADH1, encoding pyruvate decarboxylase and ethanol dehydrogenase, respectively, were also the V profile, indicating that ethanol production was co-regulated by ethanol gradient in the continuous VHG ethanol fermentation.The concentrations of AKG and SUC in the clockwise-branch of the TCA cycle showed the invert S profile, but those of ACCOA, OAA, FUM and MAL in the counter clock branch exhibit the N profile, synchronizing with that for FBP, GAP and PYR in the glycolytic pathway, indicating that the TCA cycle in yeast cells was incomplete during the continuous VHG ethanol fermentation, and that the counter clockwise branch was the main flux direction The concentration of extracellular CIT fluctuated in the range of10-20μmol/g(DCW), which was much higher than those observed with other metabolites in the TCA cycle. Moreover, the expression level of CIT2, encoding peroxisomal citrate synthase, was much higher than that of CIT1, encoding mitochondrial citrate synthase, revealing that glyoxylate cycle worked in yeast cells during the continuous VHG ethanol fermentation. During continuous VHG ethanol fermentation, the average concentration of extracellular glycerol was as high as11.5g/L. The expression level of GPD1, encoding glycerol-3-phosphate dehydrogenase associated with osmoregulation, was much higher than that of GPD2encoding glycerol-3-phosphate dehydrogenase involved in the redox balance, which also synchronized with the profile of residual glucose, suggesting that glycerol in yeast cells was synthesized mainly for osmoregulation. The concentration of intracellular TRE reached40-80μmol/g(DCW), which synchronized with the profile of ethanol production. The expression levels of genes associated with trehalose synthesis were much higher than those in glycogen synthesis, indicating that trehalose synthesis was mainly for ethanol tolerance in the continuous VHG ethanol fermentation.Transcriptomic analysis showed that the expression levels of genes associated with the syntheses of ribosomal proteins, nucleotides, amino acids, coenzymes, cell membranes and walls oscillated sequentially during the process oscillation, and significant phase shifts with respect to the profiles of intracellular metabolites were observed. Therefore, the oscillatory behavior of yeast cells under continuous VHG ethanol fermentation conditions not only was reflected in the process parameters but also in the levels of gene expression.