Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)


Agricultural and Biological Engineering

Committee Chair

Michael Ladisch

Committee Member 1

Nathan Mosier

Committee Member 2

Eduardo Ximenes

Committee Member 3

Clint Chapple


An important hindrance to commercialization of lignocellulosic ethanol is the high cost of enzymes. Reducing enzyme loadings is necessary to reduce costs. Knowledge of the inhibitors of these enzymes is necessary to better inform enzyme development and process design. Five factors have been identified: soluble inhibitors, solid lignin adsorption, product inhibition, mixing efficiency and oxygen deactivation of the enzymes that hydrolyze cellulose to glucose. These factors are intertwined and properly assessing them individually require the elimination of the others. Isolating each factor, however, has not been done throughout literature leading to lumped constants. Soluble inhibitors reduce conversion sharply leading to high enzyme loadings and impeding the evaluation of any of the other factors. Through washing, the soluble inhibitors may be eliminated, and only washed biomass (either sugar cane bagasse or corn stover) was used to study the other factors. This work further investigates adsorption on lignin, mixing, and the effect of air on washed pretreated sugarcane bagasse and corn stover.

Studies of enzyme adsorption on lignin, showed lignin/enzyme interaction was temperature dependent and proportional to pretreatment severity. Lowering reaction temperatures to 30°C, eliminated enzyme adsorption and was opposite to what was expected, indicating a possible entropic process. On a practical basis, the additional free enzyme partial makes up for the lower activity of the enzyme mixture that occurs due to reduced reaction rate at the lower temperature of 30°C. Lower hydrolysis rates also require longer reaction times to achieve the same extent of conversion to glucose.

An alternative to counter adsorption of enzyme on lignin occurs by regulating the amount of lignin exposed by adjusting pretreatment conditions. At higher temperatures a large portion of lignin is solubilized and redeposited, increasing lignin exposure and adsorption is higher. At a lower severity, lignin is less exposed, and adsorption is lower. However, higher severity is needed to increase the accessibility of cellulose, thereby facilitating accessibility and conversion. For sugarcane bagasse a 10.74 severity pretreatment (200°C for 20 minute) using liquid hot water resulted in minimal protein adsorption and therefore was interpreted to coincide with a small extent of lignin exposure, as qualitatively confirmed using SEM. Efficient conversion (71- 76%) was achieved when hydrolysis with 6.5 mg of Cellic CTEC3 / g total solids. A more recalcitrant biomass would require a more intense pretreatment to be hydrolyzed at satisfactory levels.

Mixing and product inhibition were more intricately linked than the others. When efficient mixing was achieved, product inhibition was decreased relative to cases where mixing was not readily achieved. In these runs, concentrations of pretreated and washed corn stover were at initial concentrations of 10 to 200 g/L. At 200 g/L, the higher efficiency led to faster liquefaction of biomass in the early stages. Faster liquefaction resulted in significantly high glucose conversions (up to 47% final yields) after 72 hours of hydrolysis compared to minimally liquefied material where conversion was 34%

Efficient mixing allowed deactivation due to air to be evaluated properly. This factor is the least understood in the literature and has a potentially major effect on the amount enzyme required for a given level of hydrolysis. Deactivation was isolated and observed by measuring conversion in a mixed 1 L reactor either in the presence of absence of air, except in these experiments with a different enzyme formulation, Cellic CTEC2 was used at 3.6 mg protein (Cellic CTEC2) / g solids. Cellic CTEC2 has lower activity, and the lower amount ensured that differences between the two conditions would be more obvious. Air was shown to decrease conversion by 10 to 15% with lower loss of activity corresponding to high solids loading.

The impact of unfavorable conditions (presence of lignin, inefficient mixing and inadequate air exposure) can be minimized by adjusting the biomass pretreatment and hydrolysis processes. The extent of adsorption of cellulolytic enzymes on lignin can be reduced by lowering hydrolysis temperature or reducing pretreatment severity. Efficient mixing facilitates liquefaction and increases final glucose conversion from cellulose compared to inefficient mixing methods. Limiting the presence of air increases enzyme activity and the associated final conversions. Adoption of the combined adjustments reduced enzyme loading by 50% (from 6 FPU to 3 FPU / g solids) for the enzyme Cellic CTEC2.