Mechanisms of lignin derived inhibition in hydrolysis of pretreated biomass at low enzyme loadings

The recalcitrance of lignocellulosic biomass materials with respect to enzyme hydrolysis is caused by structural factors and the interrelated effects of enzyme inhibitors.  While liquid hot water, dilute acid, steam explosion, ionic fluid or alkaline pretreatments result in high conversion, these are insufficient for achieving low enzyme loadings due to inhibition effects.  The products of cellulose hydrolysis - cellobiose and glucose – are known to inhibit cellulases and beta-glucosidases, with lignin-derived phenolics amplifying the overall inhibition effects.  Further, lignin exposed through pretreatment interferes with hydrolysis by adsorbing cellulases and beta-glucosidases.  The combined effects result in a conundrum:  increasing severity of pretreatment, whether by chemical addition or hydrothermal conditions, results in significantly enhanced enzyme hydrolysis  but also requires higher enzyme loadings. Excess enzymes, i.e., high enzyme loadings, are therefore needed if high yields from pretreated lignocellulosic substrates are to be achieved.  We report mechanisms by which lignin derived inhibitors negatively affect enzyme activity and show how the interactions between insoluble and soluble enzyme inhibitors mask the mechanisms involved in enzyme hydrolysis of pretreated biomass.  The identity of the inhibitors and the manner in which these molecules interact with cellulases, hemicellulases and beta-glucosidases will be discussed, together with approaches that show how enzyme loadings of 1 to 2 FPU/g total solids (after pretreatment) are sufficient to achieve 80% hydrolysis.  The current work utilizes results from our laboratory and other leading research facilities to define an integrated mechanistic framework for the complex interactions that both limit and enhance enzyme hydrolysis of cellulose.