Lignocellulosic biomass is considered as a promising alternative to fossil carbon resources. However, its chemical and structural complexity hinders lignocellulolytic enzymes progression and action inside the plant cell walls. Several studies have focused on reducing this biomass recalcitrance by disorganizing plant cell walls structures through pretreatment processes, but little is known on how enzymes dynamics and activity are affected by the biomass properties.<br/>To tackle this question, we have selected three representative biomasses (wheat straw, poplar and miscanthus) which have different composition and structural organization. 2 cm-long fragments of these biomasses were submitted to pretreatments (diluted acid, hot water, sodium chlorite and ionic liquid) chosen for their contrasted effects on the different constituents of the plant cell wall. This strategy allows us to work on a panel of 15 different biomass samples.<br/>Pretreatments efficiency was assessed by following glucose releasing during enzymatic hydrolysis over 96h. Biomass changes were also thoroughly studied by determination of the chemical composition and by spectral (fluorescence and infra-red) analysis of the different samples. Variations in cellulose, hemicellulose and lignin content were measured. In addition, structural variations were studied using NMR to determine the porosity and cellulose crystallinity, but also changes in lignin subunits linkages.<br/>Confocal fluorescence microscopy was used to assess polymers accessibility. Mobility parameters of two kinds of rhodamine-labelled probes were analyzed: polyethylene glycol probes known to interact with lignin as enzymes do, and dextran probes which are considered as inert. Probes of different hydrodynamic radii (2.3, 3.5 and 4.7nm) were used to cover the size range of the lignocellulolytic enzymes. Probes mobility measurements were performed before and after pretreatments, and also during the saccharification at different hydrolysis times in order to evaluate the impact of hydrolysis on probe mobility and therefore on substrate accessibility.<br/>Gathering all these data together has allowed to highlight the most important features affecting saccharification. In particular, correlations observed between plant cell wall modifications and probes mobility will be the framework to design a predictive model of enzymatic conversion potential that will be a useful tool to valorize lignocellulosic biomass as efficiently as possible.

Fluorescent probes dynamics to determine biomass characteristics impact on lignocellulolytic enzymes activity and mobility

253rd National Meeting (2017)

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