Decoupling Sugar Availability and Metabolic Efficiency Reveals Bottlenecks in Ethanol Production from Lignocellulosic...

Lignocellulosic hydrolysates derived from hydrothermal processing often result in low ethanol yields, which are commonly attributed to inhibitory compounds and suboptimal sugar composition. However, the relative contribution of sugar availability and microbial metabolic efficiency to these limitations remains poorly understood. This study systematically evaluated the ethanol production by Scheffersomyces stipitis using hydrolysates obtained from steam explosion (SE), subcritical water hydrolysis (SWH), enzymatic hydrolysis (EH), and their sequential combinations, under identical fermentation conditions. To decouple process limitations, ethanol yields were analyzed based on both total sugar available and sugar effectively consumed. Results showed that hydrothermal hydrolysates (SE and SWH) led to low ethanol yields (0.08-0.14 g/g), primarily due to limited sugar uptake associated with the presence of inhibitory compounds and low sugar concentrations. In contrast, enzymatic hydrolysates, particularly after SE pretreatment (SE + EH), achieved yields up to 0.50 g/g, approaching the theoretical maximum. Notably, when yields were calculated based on sugar consumed, significantly higher efficiencies were observed across all conditions, revealing that the intrinsic metabolic capacity of S. stipitis remained largely preserved even in inhibitory media. These findings demonstrate that reduced ethanol production in lignocellulosic hydrolysates is predominantly governed by limitations in sugar assimilation rather than metabolic conversion. This study provides a mechanistic framework for distinguishing between substrate accessibility and metabolic efficiency, offering new insights for the optimization of second-generation ethanol processes.