Identification and Evaluation of Low Recalcitrance Natural Populus Variants as Top Biofuels Feedstock Candidates

Recalcitrance of woody biomass governs the energy expenditure during pretreatment, as well as the loading of expensive enzymes to breakdown cellulose into glucose. Pretreatment and enzyme costs are the two largest components of the bioconversion of lignocellulosic biomass. We identified several natural Populus mutants that gave 100% higher xylan yields in low severity hydrothermal batch pretreatment and 300% higher glucan yields in enzymatic hydrolysis at lower enzyme loadings relative to the BESC Populus standard. These variants share naturally occurring mutations in a gene that is involved in regulating synthesis of lignin precursors prior to the lignin pathway. High-throughput pretreatment and co-hydrolysis showed a strong correlation between glucan and xylan yields. Variants that displayed the greatest and lowest sugar yields when pretreated at various severity conditions were hydrolyzed using cellulase only or supplemented with low to high ratios of xylanase to cellulase and also blocked with bovine serum albumin to elucidate factors responsible for recalcitrance¹. Fermentation of unpretreated lines and enzymatic hydrolysis using free fungal cellulase, showed similar trends with the high sugar yielding biomass also yielding the highest ethanol concentrations. ¹³C-¹H HSQC spectra for lignin isolated from the selected low lignin lines demonstrated that these mutants have a greater abundance of β-O-4 linkages and higher syringyl/guaiacyl ratio than the standard. Glycome profiling and flowthrough pretreatment were applied to understand structural differences in polysaccharide-lignin networks which contribute to recalcitrance. Through this study, we found that both lignin content and alterations in hemicellulose/cellulose bonding with lignin made these candidates more easily deconstructed.