(292d) Effect of Low Lignin Content in Transgenic Populus Trichocarpa On Sugar Release

We have previously shown that both lignin content and structure in natural Populus variants can affect its deconstruction by cellulases¹. Additionally, others have shown that Populus Trichocarpa (Poplar) mutant lines with low lignin content yield higher ethanol yields compared to Poplar controls^2,3. However, these studies have been at high cellulase loadings in combination with just a few pretreatment conditions, and structural features responsible for high sugars/ethanol yields have not been thoroughly investigated. Thus, in this study the effect of pretreatment type and conditions, and enzyme loading and formulation on sugar release and features responsible for high sugar/ethanol release were investigated for 22 Poplar mutants as well as controls. To optimize pretreatment conditions for highest sugar release, we first performed flowthrough pretreatment on standard poplar and then 22 Poplar mutants with reduced lignin content as well as standards, and controls.  All these materials were also subjected to high throughput pretreatment and enzymatic hydrolysis (HTPH) to screen for pretreatment conditions that gave the highest total sugar release. High throughput pretreatments were performed at hydrothermal, dilute acid, and alkaline conditions between 140 °C to 180 °C to study xylan, lignin, and total sugar release. The best performing mutants were then pretreated at the optimized conditions in batch reactors. Ultrastructure characterization of batch and flowthrough pretreated solids was done by SEM, and lignin structure was characterized by HSQC NMR. Surface properties were studied by a dye staining technique and WRV. The pretreatment liquor was analyzed for sugar release by HPLC, and the degree of polymerization (DP) of oligomeric sugars was analyzed by GPC. Enzymatic hydrolysis was performed at low to high cellulase loadings to better understand enzyme performance in cellulose deconstruction of Poplar natural mutants. Sugar release was found to increase with increasing pretreatment temperature from 140 °C to 180 °C and depended on both lignin content and lignin S/G ratio.

Abbreviations: SEM: Scanning Electron Microscopy, HSQC NMR: Heteronuclear Single Quantum Coherence Nuclear Magnetic Resonance, WRV: Water Retention Values, HPLC: High Performance Liquid Chromatography, GPC: Gel Permeation Chromatography, S/G: Syringyl/Guaiacyl Units

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2.   C. Fu, J. R. Mielenz, X. Xiao, Y. Ge, C. Y. Hamilton, M. Rodriguez, F. Chen, M. Foston, A. Ragauskas, J. Bouton, R. A. Dixon and Z.-Y. Wang, Proc. Natl. Acad. Sci., 2011, 108, 3803-3808.

3.   F. Chen and R. A. Dixon, Nat Biotech, 2007, 25, 759-761.