Cellulosic ethanol as a transportation fuel can cut greenhouse gas emissions for a sustainable earth and lower dependence on fossil fuels. However, successful penetration into an oil dominated market can only come through cost-competitiveness with gasoline, but biological conversion of cellulosic biomass to fuels is currently stymied by the need for high enzyme doses to realize commercially relevant yields for the widely pursued biological route of pretreatment followed by enzymatic hydrolysis to produce fermentable sugars. Achieving high sugar yields at low enzyme loadings can benefit from a better understanding of factors that contribute to biomass recalcitrance to deconstruction. When poplar varieties with lower lignin content due to a rare natural mutation associated with lignin biosynthesis were subjected to a high throughput pretreatment and co-hydrolysis platform, they gave higher sugar yields than standard poplar, but temperature pretreatment severity affected their rankings. In another approach to understand factors responsible for recalcitrance, application of flowthrough and batch pretreatment with dilute acid and just liquid hot water to standard poplar indicated that flowthrough pretreatment solubilized and removed 65 to 70% of the lignin before it could react further to low solubility lignin rich fragments that otherwise deposit on biomass in batch operations and hinder enzyme action. In subsequent work, combinations of enzymes and bovine serum albumin applied to solids produced by aqueous flowthrough and batch pretreatments revealed how polysaccharides in poplar were successively deconstructed layer by layer through pretreatment and subsequent hydrolysis by cellulase and xylanase combinations and through BSA addition. The standard strong acid hydrolysis procedure for measuring carbohydrate and lignin content in biomass was shown to be robust over a range of particle sizes, reaction times, and filtration strategies. Finally, elimination of shaking resulted in enzyme loadings of only 5 mg protein/g glucan able to realize nearly complete cellulose conversion, similar yields to those achieved with shaking if surfactants were added. This surprising result suggest that surfactants protect enzymes from deactivation that only occurs in shaken flasks at low enzyme to substrate ratios.