Lignocellulosic biomass is a renewable resource that can be converted to liquid fuels to reduce global dependence on fossil fuels and minimize greenhouse gas emissions. However, plants have evolved to protect their valuable cell wall polysaccharides through various mechanisms altogether termed recalcitrance. Pretreatment can overcome biomass recalcitrance and increase access to cellulose, although technologies require costly enzyme loadings to achieve high sugar yields after pretreatment, making biofuels unable to compete with petroleum-derived fuels. Recently, Co-solvent Enhanced Lignocellulosic Fractionation (CELF) has been developed as an advanced pretreatment technology to achieve high sugar yields from biomass at low enzyme loadings.
In this thesis, the reasons behind increased digestibility of Alamo switchgrass after CELF were investigated in comparison to research benchmark dilute sulfuric acid (DSA) pretreatment. CELF was found to solubilize all of the hemicellulose and nearly 80% of lignin from switchgrass. Protein quantification revealed that residual lignin in CELF solids bound negligible amounts of enzyme compared to those from DSA pretreatment. Further analysis, in conjunction with scanning electron microscopy, suggested that unlike DSA, CELF prevented lignin redeposition during pretreatment. Molecular dynamics simulations and chemical characterization techniques showed that the tetrahydrofuran (THF):water co-solvent used during CELF was responsible for unraveling of lignin structure during pretreatment and facilitating acid-catalyzed hydrolysis of lignin inter-unit linkages. CELF was also found to be capable of achieving near identical sugar yields from unmilled and milled switchgrass, unlike DSA, thus potentially eliminating an entire processing step. In subsequent work, the liquid hydrolyzate after CELF pretreatment was found to contain fermentable sugars, lignin-derived compounds and a unique sugar-derived surfactant. The surfactant was found to be highly stimulatory to aerobic and anaerobic performance of Saccharomyces cerevisiae. Lignin-derived phenolics in CELF hydrolyzate were found to be the major source of inhibition to fermenting microorganisms after removal of THF. Immiscible organic solvents were successful in extracting the majority of lignin-derived phenolics to overcome inhibition. Additionally, Saccharomyces cerevisiae was capable of acclimatizing to CELF hydrolyzate inhibitors by exposure to incremental concentrations of hydrolyzate and whole cell recycle in successive fermentation runs.