Quantifying second generation ethanol inhibition: Design of Experiments approach and kinetic model development

• Novel DoE methodology was used to guide kinetic model development.
• High levels of ethanol masked the effects of other inhibitors.
• Removal of ethanol from the DoE design enabled identification of other effects.
• A simple kinetic model accounting for DoE-identified inhibition was developed.
• DoE was also used to identify significant effects on HMF and furfural reduction.

While softwoods represent a potential feedstock for second generation ethanol production, compounds present in their hydrolysates can inhibit fermentation. In this study, a novel Design of Experiments (DoE) approach was used to identify significant inhibitory effects on Saccharomyces cerevisiae D5A for the purpose of guiding kinetic model development. Although acetic acid, furfural and 5-hydroxymethyl furfural (HMF) were present at potentially inhibitory levels, initial factorial experiments only identified ethanol as a significant rate inhibitor. It was hypothesized that high ethanol levels masked the effects of other inhibitors, and a subsequent factorial design without ethanol found significant effects for all other compounds. When these non-ethanol effects were accounted for in the kinetic model, R¯2 was significantly improved over an ethanol-inhibition only model (R¯2 = 0.80 vs. 0.76). In conclusion, when ethanol masking effects are removed, DoE is a valuable tool to identify significant non-ethanol inhibitors and guide kinetic model development.