5-06: Identifying rate limitations in the enzymatic hydrolysis of cellulose: insights from computational and experimental studies

Enzymatic hydrolysis of cellulose, a key step in the production of biofuel from lignocelluloses, is currently limited by long residence times for breaking down cellulose into glucose, especially at high cellulose conversions. The complex nature of substrate-enzyme interactions in this heterogeneous bio-catalytic process has made it difficult to determine the exact causes for rate retardation. In this work, we present: a) the use of results from experiments as well as simulations to screen the different hypotheses in literature, and b) results from model-guided experiments that quantify contributions of various rate limiting factors. Over-parameterization of models, which happens when only time-conversion data is used, is avoided by fitting our model to data from adsorption and re-suspension experiments. Specifically, we investigate the behavior of the model parameters over the conversion profile.

When product inhibition is alleviated with excess beta-glucosidase, we find completely amorphous cellulose to fit a first-order reaction curve, implying limitations only due to substrate depletion. Crystalline cellulose, however, did not fit a first-order curve and the apparent rate order varied with time. We have found that the hypotheses of crystallinity increase with conversion, and enzymes getting stuck on the cellulose surface when considered a first-order reaction, are not plausible reasons for decelerating rates. Modeling studies will be shown at two levels – 1. Micro-kinetic scale where events such as finding a reactive site, getting stuck at an obstacle, and reacting are investigated, and 2. Macroscopic scale where changes in substrate and enzyme properties as a function of conversion are incorporated.