1 Department of Chemical and Biochemical Engineering, Technical University of Denmark2 Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark
Several studies have indicated that cellulase action on cellulose fibers and their conversion to glucose is inhibited by lignin and lignin-derived phenolic substances, which are released during the pretreatment of lignocellulosic biomass. A prerequisite for optimization of the cellulose-to-glucose conversion is to either get rid of the inhibitory substances or to alter them in a way, so they no longer decrease the action of cellulases. The main focus in the present work was the investigation of the influence of the enzymes that are being expressed from the white-rot fungi when lignin was present in the cultivation broth, on the cellulase catalyzed hydrolysis of pretreated biomass, and to understand the mechanism of their action on phenolic substances. In this thesis, 44 fungi from the genus Alternaria, Fusarium, Memnoniella, Stemphylium, Ulocladium, Ganoderma, Trametes, and Polyporus were evaluated for their ability to grow on lignocellulosic material, such as sugarcane bagasse – a competitive substrate for grain bioethanol. From this investigation, four white-rot fungi (Ganoderma lucidum, Trametes versicolor, Polyporus brumalis, and Polyporus ciliatus), were selected for the growth on lignin (lignin alkaline) and investigated for production of enzymes under such conditions (Paper I). G. lucidum was found to produce high amounts of laccase which corresponded to its exceptional growth on lignocellulosic substrate and lignin. This observation led to a hypothesis that this particular laccase might act in a synergistic way with cellulase preparations and yield in higher cellulose-to-glucose catalyzed hydrolysis. To test this hypothesis the laccase-rich crude extract from G. lucidum was added to the cellulase catalyzed hydrolysis of cellulose from the pretreated sugarcane bagasse (Paper I). A positive outcome of this reaction, a 17% increase in the total glucose yields during cellulase catalyzed hydrolysis of cellulose, led to amplification of laccase gene and its expression in Pichia pastoris (Paper II). This approach was directed into obtaining a monocomponent laccase enzyme and to prove that the higher yields of cellulose-to-glucose conversion are partly due to the presence of laccase, and are not caused by the other proteins, present in the laccase-rich crude protein extract. The addition of the laccase from G. lucidum, expressed in P. pastoris resulted in a total increase in the glucose yields by 20 and 33% depending on the cellulase cocktail preparation. This discovery is significant considering the fact that the cellulase cocktail preparations, namely Cellic®CTec1 and Cellic®CTec2, are improved in respect to phenolic-derived, and end-substrate inhibitors. Additionally, the molecular dynamics simulations (MD) of the obtained amino acid sequence of the laccase from G. lucidum highlighted a potential mechanism of laccase detoxification of the cellulase-pretreated-biomass-derived inhibitors (Paper II). The mechanism of laccase reaction on the phenolic substrates was further evaluated by the literature study of the reactions that take place in the catalytic pocket of this oxidoreductases and the structural alteration that can lead to a more robust, or completely inactive, laccase (Review paper).