Insights into the action of phylogenetically diverse microbial expansins on the structure of cellulose microfibrils.

BACKGROUND: Microbial expansins (EXLXs) are non-lytic proteins homologous to plant expansins involved in plant cell wall formation. Due to their non-lytic cell wall loosening properties and potential to disaggregate cellulosic structures, there is considerable interest in exploring the ability of microbial expansins (EXLX) to assist the processing of cellulosic biomass for broader biotechnological applications. Herein, EXLXs with different modular structure and from diverse phylogenetic origin were compared in terms of ability to bind cellulosic, xylosic, and chitinous substrates, to structurally modify cellulosic fibrils, and to boost enzymatic deconstruction of hardwood pulp. RESULTS: Five heterogeneously produced EXLXs (Clavibacter michiganensis; CmiEXLX2, Dickeya aquatica; DaqEXLX1, Xanthomonas sacchari; XsaEXLX1, Nothophytophthora sp.; NspEXLX1 and Phytophthora cactorum; PcaEXLX1) were shown to bind xylan and hardwood pulp at pH 5.5 and CmiEXLX2 (harboring a family-2 carbohydrate-binding module) also bound well to crystalline cellulose. Small-angle X-ray scattering revealed a 20-25% increase in interfibrillar distance between neighboring cellulose microfibrils following treatment with CmiEXLX2, DaqEXLX1, or NspEXLX1. Correspondingly, combining xylanase with CmiEXLX2 and DaqEXLX1 increased product yield from hardwood pulp by ~ 25%, while supplementing the TrAA9A LPMO from Trichoderma reesei with CmiEXLX2, DaqEXLX1, and NspEXLX1 increased total product yield by over 35%. CONCLUSION: This direct comparison of diverse EXLXs revealed consistent impacts on interfibrillar spacing of cellulose microfibers and performance of carbohydrate-active enzymes predicted to act on fiber surfaces. These findings uncover new possibilities to employ EXLXs in the creation of value-added materials from cellulosic biomass.

Deaggregation of cellulose macrofibrils and its effect on bound water.

The purpose of this study was to determine how to control and measure the hierarchical swelling in pulp fibers via electrostatic interactions and localized osmotic pressure. A eutectic solvent system was used to systematically increase phosphate groups in the cell wall. Increase in fiber charge led to an increase in swelling properties, as expected. At a charge value around 180-200 µmol/g the macrofibrils were found to deaggregate. This led to a large jump in mesoscale swelling, from 0.9 to 2.5 mL/g, and surface area, from 400 to 1000 m2/g. This deaggregation was confirmed with X-ray scattering and solute exclusion. A novel thermoporosimetry method was used in the study. This involved splitting the nonfreezing water into two subfractions, thus allowing a more complete analysis of pore structure and surface area. The hydrated surface area for the samples was in the range 1200-1400 m2/g, which agreed well with simulations of aggregated microfibrils. Adding charge to the pulp fibers had a nonlinear effect on handsheet strength properties. This suggests that hierarchical control of fiber swelling may be a useful approach to improve important property pairs such as strength/density in packaging and other commercially important fiber products.

Degradation of cellulose polymorphs into glucose by HCl gas with simultaneous suppression of oxidative discoloration.

As cellulose is the main polysaccharide in biomass, its degradation into glucose is a major undertaking in research concerning biofuels and bio-based platform chemicals. Here, we show that pressurized HCl gas is able to efficiently hydrolyze fibers of different crystalline forms (polymorphs) of cellulose when the water content of the fibers is increased to 30-50 wt%. Simultaneously, the harmful formation of strongly chromophoric humins can be suppressed by a simple addition of chlorite into the reaction system. 50-70 % glucose yields were obtained from cellulose I and II polymorphs while >90 % monosaccharide conversion was acquired from cellulose IIIII after a mild post-hydrolysis step. Purification of the products is relatively unproblematic from a gas-solid mixture, and a gaseous catalyst is easier to recycle than the aqueous counterpart. The results lay down a basis for future practical solutions in cellulose hydrolysis where side reactions are controlled, conversion rates are efficient, and the recovery of products and reagents is effortless.

Nanoscale Mechanism of Moisture-Induced Swelling in Wood Microfibril Bundles.

Understanding nanoscale moisture interactions is fundamental to most applications of wood, including cellulosic nanomaterials with tailored properties. By combining X-ray scattering experiments with molecular simulations and taking advantage of computed scattering, we studied the moisture-induced changes in cellulose microfibril bundles of softwood secondary cell walls. Our models reproduced the most important experimentally observed changes in diffraction peak locations and widths and gave new insights into their interpretation. We found that changes in the packing of microfibrils dominate at moisture contents above 10-15%, whereas deformations in cellulose crystallites take place closer to the dry state. Fibrillar aggregation is a significant source of drying-related changes in the interior of the microfibrils. Our results corroborate the fundamental role of nanoscale phenomena in the swelling behavior and properties of wood-based materials and promote their utilization in nanomaterials development. Simulation-assisted scattering analysis proved an efficient tool for advancing the nanoscale characterization of cellulosic materials.

Experimental and Simulation Study of the Solvent Effects on the Intrinsic Properties of Spherical Lignin Nanoparticles.

Spherical lignin nanoparticles (LNPs) fabricated via nanoprecipitation of dissolved lignin are among the most attractive biomass-derived nanomaterials. Despite various studies exploring the methods to improve the uniformity of LNPs or seeking more application opportunities for LNPs, little attention has been given to the fundamental aspects of the solvent effects on the intrinsic properties of LNPs. In this study, we employed a variety of experimental techniques and molecular dynamics (MD) simulations to investigate the solvent effects on the intrinsic properties of LNPs. The LNPs were prepared from softwood Kraft lignin (SKL) using the binary solvents of aqueous acetone or aqueous tetrahydrofuran (THF) via nanoprecipitation. The internal morphology, porosity, and mechanical properties of the LNPs were analyzed with electron tomography (ET), small-angle X-ray scattering (SAXS), atomic force microscopy (AFM), and intermodulation AFM (ImAFM). We found that aqueous acetone resulted in smaller LNPs with higher uniformity compared to aqueous THF, mainly ascribing to stronger solvent-lignin interactions as suggested by MD simulation results and confirmed with aqueous 1,4-dioxane (DXN) and aqueous dimethyl sulfoxide (DMSO). More importantly, we report that both LNPs were compact particles with relatively homogeneous density distribution and very low porosity in the internal structure. The stiffness of the particles was independent of the size, and the Young's modulus was in the range of 0.3-4 GPa. Overall, the fundamental understandings of LNPs gained in this study are essential for the design of LNPs with optimal performance in applications.