Dynamical assessment of fluorescent probes mobility in poplar cell walls reveals nanopores govern saccharification

Abstract

BackgroundImproving lignocellulolytic enzymes’ diffusion and accessibility to their substrate in the plant cell walls is recognised as a critical issue for optimising saccharification. Although many chemical features are considered as detrimental to saccharification, enzymes’ dynamics within the cell walls remains poorly explored and understood. To address this issue, poplar fragments were submitted to hot water and ionic liquid pretreatments selected for their contrasted effects on both the structure and composition of lignocellulose. In addition to chemical composition and porosity analyses, the diffusion of polyethylene glycol probes of different sizes was measured at three different time points during the saccharification.ResultsProbes’ diffusion was mainly affected by probes size and pretreatments but only slightly by saccharification time. This means that, despite the removal of polysaccharides during saccharification, diffusion of probes was not improved since they became hindered by changes in lignin conformation, whose relative amount increased over time. Porosity measurements showed that probes’ diffusion was highly correlated with the amount of pores having a diameter at least five times the size of the probes. Testing the relationship with saccharification demonstrated that accessibility of 1.3–1.7-nm radius probes measured by FRAP on non-hydrolysed samples was highly correlated with poplar digestibility together with the measurement of initial porosity on the range 5–20 nm.ConclusionMobility measurements performed before hydrolysis can serve to explain and even predict saccharification with accuracy. The discrepancy observed between probes’ size and pores’ diameters to explain accessibility is likely due to biomass features such as lignin content and composition that prevent probes’ diffusion through non-specific interactions probably leading to pores’ entanglements.

Three-Dimensional Imaging of Plant Cell Wall Deconstruction Using Fluorescence Confocal Microscopy

“…However, the valorization of LB remains a challenge because of its complex structure and chemical composition, which makes it naturally recalcitrant to enzymatic degradation [4]. Despite extensive research on identifying the chemical and structural parameters underlying LB recalcitrance, such as lignin content [5], cellulose crystallinity [6], degree of polymerization [7] and porosity [8], the identified parameters are not universal, but rather, specific to the biomass species and pretreatment type. Moreover, structural parameters at cellular and tissular scale have not been investigated.…”

Tracking of enzymatic biomass deconstruction by fungal secretomes highlights markers of lignocellulose recalcitrance

“…Also, since water sorption was also strongly correlated to the particle size, this reinforces the fact that reducing particle size is a key step to optimise hydrolysis [51]. Overall, among all the markers shown to be strongly correlated to enzymatic hydrolysis yield [47, 51–56], water sorption, even not the fastest one to measure, seems representative of the factors guiding hydrolysis.…”

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

“…To demonstrate the sFLIM capability to dissect molecular interactions between lignin and fluorescently-tagged molecules, we first used three different samples (Figure 3): native wheat straw (WS), native wheat straw incubated with PEG tagged with rhodamine (PR10), and native wheat straw with dextran tagged with rhodamine (DR10). PR10 is known to interact with lignin while DR10 is supposed to be inert 13,14,15 . sFLIM curves (Figure 3, top) show some modifications that can be achieved between the reference sample (WS) and two interaction cases (DR10 and PR10).…”