S102: Directed evolution and adaptation to generate Saccharomyces strains able to ferment pretreated pine at high solids concentrations and to display resistance to inhibitory compounds

Softwood is a major source of lignocellulosic biomass, however, pretreatment creates potentially inhibitory compounds including furfural, 5-hydroxymethylfurfural, levulinic acid, acetic acid, formic acid, uronic acid, 4-hydroxybenzoicacid, vanillic acid, vanillin, phenol, cinnamaldehyde, formaldehyde, 2-furoic acid, 3,4-dihydroxybenzoic acid, 3,4-dihydroxybenzaldehyde, benzoic acid, and ferulic acid. Using commercially available yeasts for ethanol production from pretreated pine often results in poor yields under conditions of solids loading ≥ 10% w/v.  Directed evolution and adaptation was used to generate Saccharomyces yeast strains capable of fermenting pretreated pine at 17.5% w/v solids loading.  One strain, designated AJP50, routinely reached maximum theoretical yields in simultaneous saccharification and fermentation experiments using pretreated pine, 15 FPU/g d wt cellulases, and a short adaptation phase at  lower solids (7% w/v) loading prior to inoculation into high solids. Isolated colonies from AJP50 fermentations were screened for their ability to grow in the presence of inhibitory compounds and to directly ferment pretreated pine solids at 17.5% w/v.  All isolates performed well, reaching over 95% of the maximum theoretical yield by 72 h fermentation when the seed culture was produced using media containing inhibitory compounds.  When seed cultures were prepared without inhibitory compounds, performance of some of the isolates placed directly into high solids was poor.  Two isolates reached over 95% of the maximum theoretical yield of ethanol by 72h of fermentation regardless of the presence of inhibitory compounds in the seed culture. This represents the first successful direct inoculation into high solids using pretreated pine as the substrate.