Fluorescence techniques can reveal cell wall organization and predict saccharification in pretreated wood biomass

The conversion of cell wall biomass to sugar for fermentation to ethanol requires chemical or physical pre-treatments to disrupt the recalcitrant plant cell walls and to make the cellulose accessible to cellulolytic enzymes. Multiscale study of biomass deconstruction gives access to key insights into the cell wall and lignin changes induced by pretreatment. Few studies have compared the effect of pretreatment of different biomass species on the cell wall accessibility. Considering representative softwood (pine) and hardwood (poplar) biomass, we studied the impact of two pretreatments on enzymatic saccharification and cell wall structure and accessibility. Lignin fluorescence properties were investigated by measuring fluorescence lifetime in addition to chemical analysis, and the accessibility of biomass was assessed using fluorescent probes consisting of rhodamine labeled polyethylene glycol (PEG) molecules ranging from 10 to 40 kDa. Hot water treatment and chlorite delignification altered chemical structure and fluorescence lifetime, which was positively correlated with glucose conversion and negatively correlated with lignin and beta-O-4' contents. Imaging distribution of the probes indicated that chlorite pretreatment resulted in a more uniform distribution of probe in the cell wall compared to hot water treatment. The interaction between cell wall and fluorescent PEG probes was evaluated using Forster Resonance Energy Transfer (FRET) and fluorescence microscopy. The FRET efficiency showed a high negative correlation with the probe size and was greatly increased by chlorite delignification, reflecting increased accessibility to the probe and interaction. Thus the accessibility and interactions of small probes in pretreated biomass could be a relevant indicator of potential for saccharification, whereas fluorescence lifetime provides a new criteria for assessing relevant cell wall structural modifications related to enzymatic conversion of lignocellulosic biomass.