Enzyme and Microbial Technology 48 (2011) 408–415 Contents lists available at ScienceDirect Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass Youngmi Kim a,b , Eduardo Ximenes a,b , Nathan S. Mosier a,b , Michael R. Ladisch a,b,c,∗ a Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, IN 47907-2022, United States b Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN 47907-2022, United States c Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907-2022, United States article info Article history: Received 7 December 2010 Received in revised form 28 January 2011 Accepted 31 January 2011 Keywords: Cellulase Inhibition Phenolic compounds Deactivation Liquid hot water pretreatment LHW Lignocellulose abstract 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 (hydrox- ymethyl 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 pre- treatment 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 25 mg 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. © 2011 Elsevier Inc. All rights reserved. 1. Introduction Liquid hot water, steam explosion, and dilute acid pretreatments generate soluble inhibitors which hamper enzymatic hydrolysis as well as fermentation of sugars to ethanol [1–6]. Toxic and inhibitory compounds will vary with pretreatment and include soluble sug- ars, furan derivatives (hydroxymethyl fulfural, furfural), organic acids (acetic, formic and, levulinic acid), and phenolic compounds [3,5–7]. The amounts of these soluble inhibitors and their distribu- tion depend on type and severity of pretreatment, concentration of lignocellulosic solids during pretreatment and hydrolysis, and biomass type. They become more pronounced as the biomass con- centration in the hydrolysis slurry increases [1,2,8,9]. Washing the pretreated solids with hot water improved enzymatic digestibil- ity of various pretreated lignocellulose feedstocks, thus indicating that at least some of the formed inhibitors are water soluble [2,3,8–11]. ∗ Corresponding author at: Laboratory of Renewable Resources Engineering, Pur- due University, West Lafayette, IN 47907-2022, United States. Tel.: +1 765 494 7022; fax: +1 765 494 7023. E-mail address: ladisch@purdue.edu (M.R. Ladisch). Phenolic acids, and particularly, tannic and gallic acid, inhibit -glucosidase from Trichoderma reesei about twice as much as -glucosidase from Aspergillus niger and cause deactivation as well as inhibition [5,6]. Phenol concentrations that result in pronounced decreases in enzyme activity correspond to soluble components associated with 50 g/L or more of pretreated wood. Similar obser- vations have been reported for acid pretreated corn stover [12]. The action of complex mixtures of phenolic compounds has resulted in diverse conclusions regarding their effect on cellulases [3,5–7,13,14]. The purpose of this study was to identify major soluble inhibitors released during LHW (liquid hot water) pretreatment and to assess the extent of inhibition in high-solids pretreatment slurries. The effect of removing individual inhibitors from pre- treatment liquid on cellulose hydrolysis was examined. Inhibitory effects of phenolic compounds were also examined by removing soluble phenolics from pretreatment liquid using Pluronic L62D, activated carbon, or ethyl acetate, recovering the inhibitors, and then adding them back to cellulase in measured amounts. The impact of different inhibitor fractions, and the manner in which they inhibit or precipitate cellulase proteins was determined. This study gave insights into the contribution of the identified inhibitors on enzyme inhibition and deactivation as well as approaches for 0141-0229/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2011.01.007