Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass.

Liquid hot water, steam explosion, and dilute acid pretreatments of lignocellulose generate soluble inhibitors which hamper enzymatic hydrolysis as well as fermentation of sugars to ethanol. Toxic and inhibitory compounds will vary with pretreatment and include soluble sugars, furan derivatives (hydroxymethyl fulfural, furfural), organic acids (acetic, formic and, levulinic acid), and phenolic compounds. Their effect is seen when an increase in the concentration of pretreated biomass in a hydrolysis slurry results in decreased cellulose conversion, even though the ratio of enzyme to cellulose is kept constant. We used lignin-free cellulose, Solka Floc, combined with mixtures of soluble components released during pretreatment of wood, to prove that the decrease in the rate and extent of cellulose hydrolysis is due to a combination of enzyme inhibition and deactivation. The causative agents were extracted from wood pretreatment liquid using PEG surfactant, activated charcoal or ethyl acetate and then desorbed, recovered, and added back to a mixture of enzyme and cellulose. At enzyme loadings of either 1 or 25mg protein/g glucan, the most inhibitory components, later identified as phenolics, decreased the rate and extent of cellulose hydrolysis by half due to both inhibition and precipitation of the enzymes. Full enzyme activity occurred when the phenols were removed. Hence detoxification of pretreated woods through phenol removal is expected to reduce enzyme loadings, and therefore reduce enzyme costs, for a given level of cellulose conversion.

Enzyme hydrolysis and ethanol fermentation of liquid hot water and AFEX pretreated distillers' grains at high-solids loadings.

The dry milling ethanol industry produces distiller's grains as major co-products, which are composed of unhydrolyzed and unfermented polymeric sugars. Utilization of the distiller's grains as an additional source of fermentable sugars has the potential to increase overall ethanol yields in current dry grind processes. In this study, controlled pH liquid hot water pretreatment (LHW) and ammonia fiber expansion (AFEX) treatment have been applied to enhance enzymatic digestibility of the distiller's grains. Both pretreatment methods significantly increased the hydrolysis rate of distiller's dried grains with solubles (DDGS) over unpretreated material, resulting in 90% cellulose conversion to glucose within 24h of hydrolysis at an enzyme loading of 15FPU cellulase and 40 IU beta-glucosidase per gram of glucan and a solids loading of 5% DDGS. Hydrolysis of the pretreated wet distiller's grains at 13-15% (wt of dry distiller's grains per wt of total mixture) solids loading at the same enzyme reduced cellulose conversion to 70% and increased conversion time to 72h for both LHW and AFEX pretreatments. However, when the cellulase was supplemented with xylanase and feruloyl esterase, the pretreated wet distiller's grains at 15% or 20% solids (w/w) gave 80% glucose and 50% xylose yields. The rationale for supplementation of cellulases with non-cellulolytic enzymes is given by Dien et al., later in this journal volume. Fermentation of the hydrolyzed wet distiller's grains by glucose fermenting Saccharomyces cerevisiae ATCC 4124 strain resulted in 100% theoretical ethanol yields for both LHW and AFEX pretreated wet distiller's grains. The solids remaining after fermentation had significantly higher protein content and are representative of a protein-enhanced wet DG that would result in enhanced DDGS. Enhanced DDGS refers to the solid product of a modified dry grind process in which the distiller's grains are recycled and processed further to extract the unutilized polymeric sugars. Compositional changes of the laboratory generated enhanced DDGS are also presented and discussed.