To reduce the onset and impact of climate change, it is necessary to drastically reduce theconsumption of fossil fuels. Biofuels and chemicals made from lignocellulosic biomass havebeen proposed as a promising alternative to fossil fuels and chemicals. However, productioncosts are currently higher for bio-based products than for the production of similar fossil basedfuels and chemicals. The higher conversion costs are due primarily to the inherent resistance oflignocellulosic biomass to microbial or chemical degradation, commonly referred to as biomassrecalcitrance. More cost effective and sustainable processes must be developed to convertlignocellulosic biomass to fuels and chemicals, and this requires both a better understanding ofbiomass recalcitrance and the conversion process. One approach to improving processeconomics is to increase the substrate (solids) concentration during the process, which increasesproduct concentration and reduces separation costs. However, this has been shown to decreasecellulose conversion yields. It is thus necessary to better understand this high solids effect, andto determine if it can be reduced by modification of the biomass.This thesis explores the relationship between water and lignocellulosic biomass, and seeks tounderstand how biomass-water interactions are related to and affect biomass recalcitrance.Furthermore, it explores how the high solids effect is related to biomass-water interactions, andhow these interactions also impact solid-liquid separation processes during biomass conversion.In this thesis, a variety of pretreated biomaterials were evaluated for both their level ofrecalcitrance, as measured by enzymatic hydrolysis with commercial cellulase enzymes, and forthe amount and types of biomass-water interactions, using Water Retention Value (WRV)and 1H time domain Low Field Nuclear Magnetic Resonance (LFNMR) relaxometry methods.As well, two dimensional (2D) T1T2 NMR, was applied for the first time to pretreated biomass,and allowed for the identification of different pools of water in the biomass, and to measure theeffect of solutes on biomass morphology. Enzymatic hydrolysis experiments were carried out atdifferent solids concentrations, and were compared to changes in biomass-water interactions tobetter understand their relationship to the high solids effect. Finally, biomass-water interactionswere monitored during the course of enzymatic hydrolysis of pretreated biomass, and auxiliaryenzymes were used to attempt to improve solid-liquid separation after hydrolysis.11It was found that increased water retention and constraint by pretreated biomass correlated toincreased cellulose conversion yields for a wide variety of pretreated materials from differentpretreatment processes and severities. This suggests that biomass-water interactions may begenerally related to biomass recalcitrance. T1T2 NMR showed significant changes to biomasswaterinteractions after pretreatment, with cell wall water becoming much less constrained.Results also indicated that voids within the biomass increased in volume when subjected to watercontaining enzyme sized proteins, which was interpreted as an osmosis effect. Similarly,preliminary results from T1T2 NMR analysis indicated that this method was capable of detectingsmall changes to the surface chemistry of cellulose after oxidation with Lytic PolysaccharideMono-Oxygenase enzymes. The high solids effect was found to be related to the disappearanceof free water from the system, i.e. water only interacting weakly with the biomass, and to acollapsing of pores in the biomass with increased solids concentrations, as well as decreaseddiffusion rates for water in the biomass matrix. The high solids effect could be reduced bymodifying the pretreated biomass, suggesting that this effect can be reduced through tailoreddesign of pretreatment methods. WRV and water constraint was found to increase during thecourse of enzymatic hydrolysis, most likely due to decreasing particle sizes. This suggests thatsolid liquid separations are more difficult after enzymatic hydrolysis. No improvement to solidliquid separation properties were found by the addition of laccase to the standard cellulasemixture during hydrolysis.This thesis has shown that biomass-water interactions play an important role in lignocellulosicbiomass conversion, and they have an underlying relationship to biomass recalcitrance. Themethods developed in this thesis can be used both to better understand these interactions, but arealso to some extent capable of predicting the biomass recalcitrance of pretreated materials. Thismay have industrial applications for improving pretreatment processes and process monitoring.The high solids effect remains a challenge for improving process economics; however this workhas provided important clues about the role of water in causing this effect, and to howmodifications of the biomass during pretreatment could produce biomass which is lessrecalcitrant at high solids concentrations.