High-cell density two-stage self-cycling fermentation system for the enhanced production of ethanol from steam-exploded poplar hydrolysate

The economics of lignocellulosic ethanol production by Saccharomyces cerevisiae remain a challenge due to low product yields, which can be partially addressed by improving the performance of pretreatment optimization and fermentation systems, an aspect that has received limited attention. In this study, we investigated how the combination of self-cycling fermentation (SCF) with continuous adapted feeding (controlled by evolved gas) in a two-stage system could improve bioethanol production from steam-exploded poplar hydrolysates. This system benefited from the improved productivity associated with SCF and from the high titers obtained in fed-batch operation. The system consisted of a first fed-batch SCF stage, which led to approximately two-fold improvements in cell dry weight compared to conventional batch culture and low residual glucose (< 0.5 g/L), demonstrating efficient substrate utilization by the yeast. The second high-cell density SCF stage was initiated upon reaching a metabolic threshold by adding a pulse feed in the harvested medium to further enhance ethanol titer and productivity. The patterns of glucose consumption, ethanol production, and evolved gas flow rate were all reproducible between the SCF cycles. The two-stage fermentation approach led to final ethanol titer of ~ 11% (v/v) with an overall yield of 0.453 g/g and productivity of 1.33 g/L/h (a 30% improvement over the first stage). Overall, this study presents a robust two-stage high-cell density SCF system for lignocellulosic ethanol production and highlights the feasibility and potential of implementing it in biorefinery processes.