Identification of developmental stage and anatomical fraction contributions to cell wall recalcitrance in switchgrass

Jacob D. Crowe, Nicholas Feringa, Sivakumar Pattathil, Brian Merritt, Cliff Foster, Dayna Dines, Rebecca G. Ong, David B. Hodge

Heterogeneity within herbaceous biomass can present important challenges for processing feedstocks to cellulosic biofuels. Alterations to cell wall composition and organization during plant growth represent major contributions to heterogeneity within a single species or cultivar. To address this challenge, the focus of this study was to characterize the relationship between composition and properties of the plant cell wall and cell wall response to deconstruction by NaOH pretreatment and enzymatic hydrolysis for anatomical fractions (stem internodes, leaf sheaths, and leaf blades) within switchgrass at various tissue maturities as assessed by differing internode. Substantial differences in both cell wall composition and response to deconstruction were observed as a function of anatomical fraction and tissue maturity. Notably, lignin content increased with tissue maturity concurrently with decreasing ferulate content across all three anatomical fractions. Stem internodes exhibited the highest lignin content as well as the lowest hydrolysis yields, which were inversely correlated to lignin content. Confocal microscopy was used to demonstrate that removal of cell wall aromatics (i.e., lignins and hydroxycinnamates) by NaOH pretreatment was non-uniform across diverse cell types. Non-cellulosic polysaccharides were linked to differences in cell wall response to deconstruction in lower lignin fractions. Specifically, leaf sheath and leaf blade were found to have higher contents of substituted glucuronoarabinoxylans and pectic polysaccharides. Glycome profiling demonstrated that xylan and pectic polysaccharide extractability varied with stem internode maturity, with more mature internodes requiring harsher chemical extractions to remove comparable glycan abundances relative to less mature internodes. While enzymatic hydrolysis was performed on extractives-free biomass, extractible sugars (i.e., starch and sucrose) comprised a significant portion of total dry weight particularly in stem internodes, and may provide an opportunity for recovery during processing. Cell wall structural differences within a single plant can play a significant role in feedstock properties and have the potential to be exploited for improving biomass processability during a biorefining process. The results from this work demonstrate that cell wall lignin content, while generally exhibiting a negative correlation with enzymatic hydrolysis yields, is not the sole contributor to cell wall recalcitrance across diverse anatomical fractions within switchgrass.