The inhibitory role of lignin in the enzymatic hydrolysis of softwoods

Ethanol from biomass is one of the most promising technologies for the production of a renewable and environment friendly liquid biofuel. Currently, industrial production of ethanol is mostly based on sugar or starch-based substrates. However, ethanol produced through the bioconversion of lignocellulosic substrates such as woody and agricultural residues by enzymatic hydrolysis and fermentation, is a promising way of producing a green fuel. Softwoods are characterized by a high lignin content, which makes their enzymatic hydrolysis and eventually conversion to ethanol extremely difficult. Pretreatment of softwoods makes the substrate more accessible to cellulose-degrading enzymes (cellulases) and facilitates the cellulose degradation by modifying the physicochemical properties of the substrate. Different studies have shown that the ability of cellulases to hydrolyze pretreated softwoods is limited by various substrate factors such as, porosity, cellulose crystallinity, available surface area and the physicochemical characteristics of the residual lignin. Although the influence of cellulose properties on the enzymatic hydrolysis of lignocellulose has been studied extensively, the role of lignin as a substrate limiting factor has been more difficult to elucidate. This dissertation has focused on aspects relevant to the improvement of the enzymatic hydrolysis of softwood substrates. The first series of experiments were designed to address the possible inhibitory role of lignin in the enzymatic hydrolysis of lignocelluloses. A quantitative approach to compare the inhibition ability of lignin with other classical cellulase inhibitors was developed and assessed. In a related series of experiments, novel cellulase complexes, characterized by their higher hydrolytic ability on lignocellulosic substrates were also evaluated. A quantitative evaluation of the impact of lignin on the hydrolytic ability of various carbohydrases was performed. The study demonstrated that the magnitude of the lignin inhibition, on a concentration basis, was comparable to that of classical cellulase inhibitors. The inhibition by lignin followed a mixed-type pattern (competitive or uncompetitive, depending on the enzyme and substrate assayed). The second part of the research showed that a novel Penicillium sp. cellulase complex was more effective in the enzymatic hydrolysis of the pretreated softwood than commercially available cellulases. It was apparent that the Penicillium sp. cellulases yielded up to 2.5-fold more glucose from softwood substrates than was obtained when hydrolysis was carried out using Trichoderma sp. enzymes. Thus, a novel enzyme complex with a particularly high hydrolytic ability was identified and its application to the hydrolysis of pretreated softwood was demonstrated. Naturally occurring high levels of xylanase and β-glucosidase activities and the presence of weaker lignin-binding cellulases were postulated to be the reasons for the better performance of the Penicillium sp. enzymes. It was suggested that the increase in β-glucosidase activity reduces the cellobiose end-product inhibition, while the increase in xylanase activity increases the enzyme accessibility to cellulose by removing the shielding xylan. Weaker lignin-cellulase interactions lead to more free enzymes in solution. If this hypothesis is confirmed in the future, it could be used as a basis for further improvement of the commercially available cellulase complexes for lignocellulose bioconversion.Forestry, Faculty ofGraduat