Strong cellulase inhibitors from the hydrothermal pretreatment of wheat straw

Background: The use of the enzymatic hydrolysis of lignocellulose with subsequent fermentation to ethanol provides a green alternative for the production of transportation fuels. Because of its recalcitrant nature, the lignocellulosic biomass must be pretreated before enzymatic hydrolysis. However, the pretreatment often results in the formation of compounds that are inhibitory for the enzymes or fermenting organism. Although well recognized, little quantitative information on the inhibition of individual cellulase components by identified inhibitors is available. Results: Strong cellulase inhibitors were separated from the liquid fraction of the hydrothermal pretreatment of wheat straw. HPLC and mass-spectroscopy analyses confirmed that the inhibitors were oligosaccharides (inhibitory oligosaccharides, IOS) with a degree of polymerization from 7 to 16. The IOS are composed of a mixture of xylo- (XOS) and gluco-oligosaccharides (GOS). We propose that XOS and GOS are the fragments of the xylan backbone and mixed-linkage beta-glucans, respectively. The IOS were approximately 100 times stronger inhibitors for Trichoderma reesei cellobiohydrolases (CBHs) than cellobiose, which is one of the strongest inhibitors of these enzymes reported to date. Inhibition of endoglucanases (EGs) by IOS was weaker than that of CBHs. Most of the tested cellulases and hemicellulases were able to slowly degrade IOS and reduce the inhibitory power of the liquid fraction to some extent. The most efficient single enzyme component here was T. reesei EG TrCel7B. Although reduced by the enzyme treatment, the residual inhibitory power of IOS and the liquid fraction was strong enough to silence the major component of the T. reesei cellulase system, CBH TrCel7A. Conclusions: The cellulase inhibitors described here may be responsible for the poor yields from the enzymatic conversion of the whole slurries from lignocellulose pretreatment under conditions that do not favor complete degradation of hemicellulose. Identification of the inhibitory compounds helps to design better enzyme mixtures for their degradation and to optimize the pretreatment regimes to minimize their formation.