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Structural characterization of alkaline hydrogen peroxide pretreated grasses exhibiting diverse lignin phenotypes

Muyang Li12, Cliff Foster2, Shantanu Kelkar13, Yunqiao Pu4, Daniel Holmes5, Arthur Ragauskas467, Christopher M Saffron138 and David B Hodge1239*

Author Affiliations

1 Department of Biosystems and Agricultural Engineering, Michigan State University, Michigan, USA

2 DOE Great Lakes Bioenergy Research Center, Michigan State University, Michigan, USA

3 Department of Chemical Engineering and Materials Science, Michigan State University, Michigan, USA

4 DOE BioEnergy Science Center, Georgia Institute of Technology, Georgia, USA

5 Department of Chemistry, Michigan State University, Michigan, USA

6 Department of Chemistry and Biochemistry, Georgia Institute of Technology, Georgia, USA

7 Institute of Paper Science and Technology, Georgia Institute of Technology, Georgia, USA

8 Department of Forestry, Michigan State University, Michigan, USA

9 Department of Chemical Engineering and Materials Science, Michigan State University, Michigan, USA

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Biotechnology for Biofuels 2012, 5:38  doi:10.1186/1754-6834-5-38

Published: 6 June 2012



For cellulosic biofuels processes, suitable characterization of the lignin remaining within the cell wall and correlation of quantified properties of lignin to cell wall polysaccharide enzymatic deconstruction is underrepresented in the literature. This is particularly true for grasses which represent a number of promising bioenergy feedstocks where quantification of grass lignins is particularly problematic due to the high fraction of p-hydroxycinnamates. The main focus of this work is to use grasses with a diverse range of lignin properties, and applying multiple lignin characterization platforms, attempt to correlate the differences in these lignin properties to the susceptibility to alkaline hydrogen peroxide (AHP) pretreatment and subsequent enzymatic deconstruction.


We were able to determine that the enzymatic hydrolysis of cellulose to to glucose (i.e. digestibility) of four grasses with relatively diverse lignin phenotypes could be correlated to total lignin content and the content of p-hydroxycinnamates, while S/G ratios did not appear to contribute to the enzymatic digestibility or delignification. The lignins of the brown midrib corn stovers tested were significantly more condensed than a typical commercial corn stover and a significant finding was that pretreatment with alkaline hydrogen peroxide increases the fraction of lignins involved in condensed linkages from 88–95% to ~99% for all the corn stovers tested, which is much more than has been reported in the literature for other pretreatments. This indicates significant scission of β-O-4 bonds by pretreatment and/or induction of lignin condensation reactions. The S/G ratios in grasses determined by analytical pyrolysis are significantly lower than values obtained using either thioacidolysis or 2DHSQC NMR due to presumed interference by ferulates.


It was found that grass cell wall polysaccharide hydrolysis by cellulolytic enzymes for grasses exhibiting a diversity of lignin structures and compositions could be linked to quantifiable changes in the composition of the cell wall and properties of the lignin including apparent content of the p-hydroxycinnamates while the limitations of S/G estimation in grasses is highlighted.

Alkaline hydrogen peroxide pretreatment; Cellulosic biofuels; Lignin; Plant cell wall analysis; Analytical pyrolysis