The doctoral dissertations of the former Helsinki University of Technology (TKK) and Aalto University Schools of Technology (CHEM, ELEC, ENG, SCI) published in electronic format are available in the electronic publications archive of Aalto University - Aaltodoc.

Role of Lignin in the Enzymatic Hydrolysis of Lignocellulose

Hetti Palonen

Dissertation for the degree of Doctor of Technology to be presented with due to permission for public examination and debate in Auditorium Komppa (Ke-2) at Helsinki University of Technology (Espoo, Finland) on the 16th of April, 2004, at 12 o'clock.

Overview in PDF format (ISBN 951-38-6272-0)   [1457 KB]
VTT Publications 520, ISSN 1455-0849

Dissertation is also available in print (ISBN 951-38-6271-2)
Copyright © 2004 VTT Technical Research Centre of Finland
VTT Publications 520, ISSN 1235-0621


Characterization, understanding and overcoming barriers of enzymatic hydrolysis of different raw materials is essential for the development of economically competitive processes based on enzymatic treatments. This work focused on factors relevant for the improvement of enzymatic hydrolysis of lignocellulose raw materials derived from softwood. The major interest of the work was in lignin. Specific areas addressed were the role of lignin in the unproductive binding of cellulases, which restricts the hydrolysis of cellulose, and enzymatic modification of lignin in order to improve cellulose hydrolysis. In addition, suitability a new pretreatment method, wet oxidation, was evaluated for softwood.

The binding of Trichoderma reesei CBH I and CBH II enzymes on bacterial microcrystalline cellulose (BMCC) was shown to be determined by a co-operative effect of the two domains, the cellulose binding domain (CBD) and the catalytic domain (CD). Binding of the intact CBH I on bacterial microcrystalline cellulose (BMCC) was fully reversible, while the binding of CBH II was only partly reversible. The cellulases CBH I and EG II were adsorbed on steam pretreated softwood (SPS) and lignin. The observation that the presence of CBD clearly enhanced the binding of the enzymes on SPS and especially on lignin, suggests that unspecific adsorption is dominated by the affinity of the CBD.

The wet oxidation pretreatment studies gave information on the importance of substrate structure in the enzymatic hydrolysis. This pretreatment method was applied to softwood for the first time. In the wet oxidation pretreatment studies, the total recovery of carbohydrates was high and the recovery of cellulose even higher than what has been reported for steam pretreated softwood. Lignin fraction of the substrate remained mainly undissolved. No clear correlation between the hydrolysis yield and lignin content could be observed. It was concluded that the location and chemical/physical structure of lignin affected the enzymatic hydrolysis more than the absolute amount of lignin. It was shown that the hydrolysis result could be improved by optimizing the pretreatment conditions, reducing the hemicellulose content or hydrolysing the residual hemicellulose by selecting a suitable combination of enzymes.

This study showed for the first time that enzymatic modification and/or removal of lignin can be combined with simultaneous cellulose hydrolysis. Both the modification of lignin surfaces by oxidative treatments with laccase alone and delignification treatment with a laccase-mediator system lead to increased hydrolysis of lignocellulose. Oxidation of lignin by laccase was achieved by the three laccases tested, produced by Trametes hirsuta, Melanocarpus albomyces and Mauginiella sp. The new laccase isolated and purified from Mauginiella sp. had enzymatic characteristics similar to many basidiomycete laccases. Different adsorption of the three laccases onto SPS did not correlate with the capability of the laccases to oxidize the substrate and consequently, to improve lignocellulose hydrolysis.

This thesis consists of an overview and of the following 5 publications:

  1. Palonen, H., Tenkanen, M. and Linder, M. 1999. Dynamic interaction of Trichoderma reesei Cellobiohydrolases Cel6A and Cel7A and cellulose at equilibrium and during hydrolysis. Appl. Environ. Microbiol. 65, pp. 5229-5233.
  2. Palonen, H., Tjerneld, F., Zacchi, G. and Tenkanen, M. 2004. Adsorption of purified Trichoderma reesei cellulases and their catalytic domains to steam pretreated softwood and isolated lignin. J. Biotechnology 107, pp. 65-72.
  3. Palonen, H., Thomsen, A.B, Tenkanen, M., Schmidt, A.S. and Viikari, L. 2004. Evaluation of wet oxidation pretreatment for enzymatic hydrolysis of softwood. Appl. Biochem. Biotechnol. Vol. 117. In press.
  4. Palonen, H., Saloheimo, M., Viikari, L. and Kruus, K. 2003. Purification, characterization and sequence analysis of a laccase from the anamorphic ascomycete Mauginiella. Enzyme Microb. Technol. 33, pp. 854-862.
  5. Palonen, H. and Viikari, L. 2004. Role of oxidative enzymatic treatments on enzymatic hydrolysis of lignocellulose. Biotechnol. Bioeng. In press.

Keywords: enzymatic hydrolysis, lignocellulose, enzymes, cellulases, Trichoderma reesei, softwood lignin, pretreatment, wet oxidation, enzymatic modification, laccase

This publication is copyrighted. You may download, display and print it for your own personal use. Commercial use is prohibited.

© 2004 Helsinki University of Technology

Last update 2011-05-26