| We report an efficient method for the study of protein-biomass and protein-lignin adsorption, based on inverse liquid chromatography, which has the potential to speed up the screening of lignin blocking molecules. This work is motivated by the lignin-blocking effect of bovine serum albumin (BSA) during enzymatic hydrolysis of pretreated lignocellulose substrates. While the use of BSA illustrated the role of lignin in preventing adsorption of some cellulase components, other lignin-blocking molecules with lower cost need to be found. Hence a method is needed that improves efficiency with which potential blocking agents might be selected, and which provides insights on the nature of interactions between blocking agents and biomass. The method developed in this dissertation is based on wet packing of pretreated and untreated biomass into liquid chromatography (LC) columns, followed by evaluation of adsorption properties determined from elution profiles from preparative scale chromatography.;In this study the performances of columns, packed under constant pressure (CP) and constant flowrate (CF), and with two different diameters were first compared. Vanillin was then tested as probe molecule to prove the concept. All the columns showed decreasing retention of vanillin with increasing column temperature, which was consistent with the Arrhenius definition of adsorption kinetics and with the results from batch experiment. Later BSA (2% w/v) was used to probe adsorption on untreated sugarcane bagasse column (SB) and pretreated corn stover (CS) column. Significant precipitation of BSA was observed on the column packed with untreated SB and operated at 50 °C and pH of 4.2 to 4.6. However, on the pretreated CS column, irreversible binding to the column was more significant than precipitation at 50 °C at 4.9 mg BSA/g CS. With the same column, we also proved that hydrolysis created larger pores thus exposing more lignin and cellulose to enzymes. This work relates the elution behavior from liquid chromatography columns, packed with lignocellulosic stationary phases, to mechanisms by which lignocellulosic materials might be better hydrolyzed through control of enzyme adsorption onto lignin. |
